Figures
Abstract
Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn’s disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain Giardia infections may attenuate PMN accumulation by decreasing the expression of the mediators responsible for their recruitment.
Citation: Cotton JA, Motta J-P, Schenck LP, Hirota SA, Beck PL, Buret AG (2014) Giardia duodenalis Infection Reduces Granulocyte Infiltration in an In Vivo Model of Bacterial Toxin-Induced Colitis and Attenuates Inflammation in Human Intestinal Tissue. PLoS ONE 9(10): e109087. https://doi.org/10.1371/journal.pone.0109087
Editor: John Wallace, University of Calgary, Canada
Received: August 18, 2014; Accepted: September 8, 2014; Published: October 7, 2014
Copyright: © 2014 Cotton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: JA Cotton is a recipient of NSERC Alexander Graham Bell Scholarship graduate student scholarship and a joint IBD studentship from Alberta Innovates Health Solutions (AIHS) and the Crohn’s and colitis foundation of Canada (CCFC). Research in AG Buret's lab is funded by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RT 690446, and the CCFC 10000008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a non-invasive, small intestinal protozoan parasite infecting a variety of animal species, including humans. Giardia duodenalis is currently subdivided into eight distinct genetic assemblages, with only assemblages A and B being infective to humans [1], [2]. Some reports suggest that these assemblages may be unique Giardia species, but much controversy remains on this topic [3], [4]. This parasite induces a response within its host that may cause malabsorptive diarrheal disease ([5]). Recent evidence also indicates that giardiasis can lead to the development of post-infectious disorders, in the intestine as well as extra-intestinally, via mechanisms that remain obscure ([6], [7]). During the acute stage of the infection, parasite loads can exceed 106 trophozoites/cm of gut but the intestinal mucosa of most Giardia-infected patients is devoid of overt signs of inflammation [8]. These observations are counter-intuitive, considering that Giardia infections increase small intestinal permeability via several mechanisms and, therefore, may indeed facilitate the translocation of luminal antigens into underlying host tissues [9]–[11]. However, microscopic duodenal inflammation has been observed in some patients with chronic assemblage B Giardia infections following metronidazole treatment [12]. Giardia assemblage B infections in vivo have been shown to induce small intestinal inflammation [13]. Therefore, it remains to be determined whether Giardia infections modulate pro-inflammatory responses within the intestinal mucosa and if these events are assemblage or isolate specific.
Transmission of Giardia infections occurs via ingestion of infectious cysts in contaminated food or water, or directly via the fecal-oral route (reviewed in [14]). This route of infection is adopted by many gastrointestinal pathogens, and as a result, Giardia co-infections may occur. Indeed, Giardia infections have been reported to occur concurrently with Ascaris sp. [15], Cryptosporidium sp. [16], Helicobacter pylori [16], [17], Vibrio cholerae [18], Salmonella sp. [19] and rotavirus [18], [20], [21]. Many of these pathogens are known to promote inflammatory responses and simultaneously induce diarrheal disease within their hosts. A recent report suggested that Tanzanian children infected with Giardia have reduced incidence rates of diarrheal disease and fever, while also displaying lower serum C-reactive protein (CRP) levels [22]. Giardia infections have also been shown to attenuate symptoms of diarrheal disease during rotavirus infection [20]. The mechanisms remain unclear, it has been suggested that Giardia infections may create an advantageous environment for other co-infecting gastrointestinal pathogens [18]. Contradictorily, a separate study suggests that Giardia infections may instead enhance symptoms of diarrheal disease during rotavirus infection [16].
Polymorphonuclear leukocytes (PMNs) are heavily involved in inflammatory responses, and essential to host defence against many bacterial and fungal pathogens. Indeed, genetic mutations or drug therapy that result in defective PMN function make individuals highly susceptible to life-threatening infections [23]–[25]. These cells have been implicated in a variety of inflammatory disorders, in the gut and beyond [26], [27]. PMN recruitment into intestinal tissues is a multistep process involving egression from bone marrow and circulation, migration through host tissues, and transepithelial migration [28]–[30]. Moreover, multiple mediators are known to promote tissue accumulation of PMNs. During acute inflammatory responses, granulocyte colony stimulating factor (G-CSF) promotes PMN bone marrow egression and increases the number of circulating PMNs [31], [32]. Production of interleukin-17A (IL-17A) by tissue-resident cells increases circulating G-CSF levels and promotes subsequent PMN bone marrow egression [32]–[34]. PMN chemokines containing a consecutive glutamate-leucine-arginine sequence (ELR+ chemokines) in their N-terminal region, such as CXCL1, CXCL2, and IL-8/CXCL8, are produced by a variety of cells within the intestinal mucosa. ELR+ chemokines are involved in multiple processes of PMN tissue recruitment, including bone marrow egression [31], exit from the blood stream [30], [35], [36], and migration through host tissues [37], [38].
Previous research has suggested that Giardia excretory/secretory products induce IL-8/CXCL8 expression in intestinal epithelial cells [39]. However, we and others have demonstrated that Giardia trophozoites attenuate IL-8/CXCL8 secretion from in vitro epithelial monolayers [40], [41]. Our research further demonstrated that Giardia cathepsin B family proteases contributed to the degradation of IL-8/CXCL8 and attenuated PMN chemotaxis [41]. As PMN accumulation contributes to the development of diarrheal disease via several different mechanisms [28], [42]–[44], we hypothesized that during a Giardia infection, the attenuation of diarrheal disease or the creation of a favourable environment for other co-infecting gastrointestinal pathogens may stem, at least partially, from Giardia’s ability to attenuate PMN recruitment. These observations support the notion that Giardia parasites may reduce host inflammatory responses. Such effects and their mechanisms have yet to be established in vivo. The present report demonstrates that Giardia infections attenuate granulocyte infiltration during acute experimental colitis induced by Clostridrium difficile toxins TcdA and TcdB, and markedly modulate the expression of several pro-inflammatory mediators, in an isolate-dependent manner. This study also shows that Giardia trophozoites decrease expression of a variety of inflammatory mediators when parasites are co-incubated with inflamed biopsy tissues from patients with Crohn’s disease. Collectively, these results indicate for the first time that certain Giardia isolates are capable of attenuating intestinal inflammatory responses in inflammatory settings in vivo and ex vivo.
Materials and Methods
Ethics statement
All studies involving human colonic mucosal biopsy tissues were approved by the Conjoint Health Research Ethics Board (CHREB) at the University of Calgary and the Calgary Health Region. In accordance with CHREB guidelines, adult subjects used in this study provided informed, written consent and a parent or guardian of any child participant provided informed, written consent on their behalf. Animal experiments were approved by the Life and Environmental Sciences Animal Care Committee of the University of Calgary, conducted in compliance with that approval, and followed guidelines established by the Canadian Council of Animal Care. All isolates used in this study have been used for over 15 years in the laboratory. Therefore, their isolation did not require their approval from an ethical review board at their time of collection.
Human biopsy tissues
Adapted from previous protocols [41], [45], intestinal biopsy tissues were collected from the descending colon of patients with active Crohn’s disease (CD). Upon collection, samples were washed once in Dulbecco’s PBS (Sigma-Aldrich) containing 0.016% 1,4-Diothioerythritol (Sigma-Aldrich) to remove loosely adherent mucous and bacteria followed by three washes with phosphate-buffered saline (PBS). Following this, biopsy tissues were placed in 96-well plates and incubated in 300 µL of OptiMEM (Life Technologies) at 37°C, 5% CO2, and 96% humidity. All studies were performed in duplicate, whereby multiple biopsy tissues were collected from each patient.
Parasites
Giardia NF trophozoites were originally obtained from a water sample during an outbreak of giardiasis in Newfoundland, Canada [46], while Giardia GS/M clone H7 trophozoites were obtained from ATCC (50581) as previously described [47]. Trophozoites were grown axenically in 15 mL polystyrene tubes (Becton-Dickinson Falcon) in Keister’s modified TYI-S-33 medium [48], [49] supplemented with piperacillin (Sigma-Aldrich) and used at peak density culture.
Giardia trophozoite isolation
Confluent tubes of Giardia NF or GS/M trophozoites were harvested by cold shock on ice for 30 minutes, pooled into 50 mL polypropylene tubes (Falcon), and centrifuged at 500×g for 10 minutes. The resulting pellets were re-suspended in a total of 10 mL of ice cold PBS (Sigma-Aldrich) and centrifuged for 10 minutes at 500×g. Pellets were then re-suspended in 3 mL of fresh PBS, trophozoites were enumerated with a hemocytometer, and adjusted to the required concentration. For ex vivo human biopsy experiments, Giardia trophozoites were adjusted to a concentration of 5.0×106 trophozoites/well, while trophozoites were adjusted to a concentration of 1.0×108 trophozoites/mL for in vivo experiments. For ex vivo experiments, cells were co-incubated with Giardia trophozoites at 37°C, 5% CO2, and 96% humidity for 6 hours.
Giardia in vivo infection
Using a previously described Giardia infection model [50], [51], male C57BL/6 mice aged 4 to 6 weeks (Charles River Laboratories, Sherbrooke, QC) were acclimatized for 1 week prior to infection with Giardia NF or GS/M trophozoites. Forty-eight hours prior to oral gavage, all mice were administered broad-spectrum antibiotics to their drinking water ad libitum (1.4 mg/mL neomycin (Alfa Aesar), 1.0 mg/mL ampicillin (Alfa Aesar) and 1.0 mg/mL vancomycin (Alfa Aesar)). This regimen was maintained for the entire duration of the study. After 48 hours, mice were administered 107 Giardia NF or GS/M trophozoites in 0.1 mL of TYI-S-33 medium or 0.1 mL TYI-S-33 medium by oral gavage. After 7 days, small intestinal parasite loads were quantified. Mice were euthanized and the first 3 cm of the small intestine distal to the ligament of Treitz were opened longitudinally, placed in 1.5 mL Eppendorf tubes containing 1 mL of PBS, and kept on ice for 15 minutes. After 15-minute incubation, tubes were vortexed, and trophozoite numbers were visually enumerated under light microscope, and total concentration was extrapolated using a hemocytometer.
Clostridium difficile TcdAB-induced colitis
Following a previously described model of C. difficile toxin-induced colitis [52], mice infected with Giardia NF or GS/M trophozoites for 7 days were intra-rectally (i.r.) administered a 100 µg solution of C. difficile A/B toxin (TcdA/TcdB) for 3 hours. Briefly, a 5F infant feeding tube catheter containing side ports (Mallinckrodt Inc.) was lubricated with a water-soluble personal lubricant and inserted 2.5 cm into the colon. With pressure applied to the anal area to prevent leakage, 100 µL of a 1.0 µg/µL TcdA/TcdB solution diluted in PBS was slowly administered over a period of 30 seconds. Following this, the tube was slowly removed and pressure was maintained for another 30 seconds. Vehicle control-treated control groups were administered PBS.
Tissue collection
Mice were euthanized via cervical dislocation 3 hours post intracolonic instillation of TcdAB or PBS. The colon was removed and samples were collected for bead-based cytokine analysis, myeloperoxidase (MPO) assays, or fixed in a fresh 4% paraformaldehyde solution for immunofluorescence, as described below. Colonic tissue samples collected for histology and immunohistochemistry were fixed in 4% paraformaldehyde solution overnight at 4°C, and, subsequently, washed and transferred to PBS at 4°C.
Cytokine analysis
Supernatants from ex vivo human biopsy tissue experiments were collected and centrifuged at 10,000 g for 15 minutes at 4°C. Resulting supernatants were decanted and stored at −70°C. Colonic tissue samples and biopsy tissues were weighed and collected in 2 mL Fast-Prep tubes (MP Biomedicals) containing a mixture of 0.9–2.0 mm stainless steel beads (NextAdvance). Tissue samples were suspended in lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Tween-20 and a Complete Minitab protease inhibitor cocktail (Roche)] at a ratio of 50 mg tissue per 1 mL lysis buffer and homogenized using a Fast-Prep24 device (MP Biochemicals) at speed 6.0 for 40 seconds. The resulting homogenate solution was collected into pyrogen-free 1.5 mL Eppendorf tubes and centrifuged at 10,000 g for 15 minutes at 4°C. Supernatants and tissue homogenate cytokine levels were assessed using a Luminex XMap assay according to manufacturer’s instructions (Luminex Corp.).
Tissue myeloperoxidase assay
Myeloperoxidase (MPO) activity was used as a marker of granulocyte infiltration [53]. Colonic tissue samples were weighed and collected in 2 mL Fast-Prep tubes (MP Biomedicals) containing a mixture of 0.9–2.0 mm stainless steel beads (NextAdvance). Tissue samples were suspended in 50 mM potassium phosphate buffer containing 5 mg/mL hexadecyltrimethylammonium bromide (Sigma-Aldrich) at a ratio of 50 mg tissue per 1 mL lysis buffer. Samples were then homogenized using a Fast-Prep24 device (MP Biochemicals) at speed 6.0 for 40 seconds. The resulting homogenate solution was collected into pyrogen-free 1.5 mL Eppendorf tubes and centrifuged at 10,000×g for 15 minutes at 4°C. Seven µL of the resulting supernatant was added to a standard 96-well plate along with 200 µL of the reaction mixture (comprised of 0.005 g O-dianisidine (Sigma-Aldrich), 30 mL of distilled H2O, 3.33 mL of potassium phosphate buffer, and 17 µL of 1% H2O2). Using a microplate scanner (SpectraMax M2e, Molecular Devices, Sunnyvale, CA), three absorbance readings at 450 nm were recorded every 30 seconds. MPO activity was measured as units of activity per milligram of tissue, with 1 unit of MPO being defined as the amount required to degrade 1 µmol of H2O2 per minute at room temperature.
Immunohistochemistry
Samples were dehydrated and embedded in paraffin wax, cut on a cryostat into 8 µm sections, and mounted on poly-D-lysine coated slides. Slides were deparaffinised and incubated in antigen retrieval solution (Tris/EDTA, pH 9.0 solution for 30 minutes at 95 degrees C. Slides were then blocked in tissue blocking buffer (1% BSA, 0.1% Triton X-100 in PBS) for 1 hour (3×20 minute washes) and incubated with an anti-MPO antibody (abcam ab9535) (1 in 200 dilution) overnight at 4°C. At room temperature, slides were washed for 1 hour (3×20 minutes), incubated for 1 hour with secondary antibody (1 in 1000), and then washed for 1 hour (3×20 minutes). After this, slides were mounted with Fluoroshield™ containing DAPI (Sigma-Aldrich) and visualized using a Leica DMR fluorescence microscope at 40× magnification.
Statistics
Parametric data are expressed as means ± standard error of measurement, while non-parametric data is expressed as a dotplots with median and range. All statistical analyses were performed using GraphPad Prism 6 software, and normality of the data was assessed prior to analysis. Parametric comparisons were made using one-way ANOVA with Tukey’s post hoc analysis. Non-parametric comparisons were made using a Mann Whitney test. Statistical significance was established at p<0.05 (*).
Results
Giardia infections attenuate granulocyte infiltration during TcdAB-induced colitis in an isolate-dependent manner
Experiments were performed to determine whether in vivo assemblage A Giardia NF or assemblage B Giardia GS/M infections were capable of attenuating acute intestinal inflammatory responses to intracolonic administration of C. difficile toxin A (TcdA) and toxin B (TcdB). This model of intestinal inflammation was selected because it induces a rapid, acute intestinal inflammatory response resulting from the administration of bacterial toxins, and Giardia infections have been reported to occur concurrently with C. difficile [16]. Small intestinal trophozoites numbers in Giardia GS/M-infected animals were higher than numbers from NF-infected animals (Figure 1A). These results are consistent with previous observations that assemblage B Giardia isolates, such as GS/M, have higher parasite burdens compared to assemblage A isolates, such as NF trophozoites [50], [54]. Administration of C. difficile TcdAB for 3 hours did not change trophozoite numbers (Figure 1A).
Male C57BL/6 mice aged 4 to 6 weeks were infected with Giardia NF or GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdAB or PBS i.r. and, after 3 hours, animals were euthanized. (A) Trophozoites counts within the upper 3 cm of the jejunum were determined. (B) Colonic myeloperoxidase (MPO) activity was determined between uninfected controls and Giardia NF-infected animals given 100 µg TcdAB or PBS. (C) Colonic MPO activity was determined between uninfected controls and Giardia GS/M-infected animals given 100 µg TcdAB or PBS. (D) Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. * p<0.05.
As previously demonstrated [52], intracolonic administration of 100 µg of C. difficile TcdAB significantly increased colonic tissue MPO activity (Figure 1B and C). As determined via immunofluorescence, numbers of MPO-positive cells increased in colonic tissues of TdcAB-trated animals compared to control animals (Figure 2). In Giardia NF-infected animals administered intracolonically with 100 µg of TcdAB, colonic MPO activity (Figure 1B) and numbers of MPO-positive cells were lower when compared against control animals (Figure 2). Contrastingly, MPO activity levels (Figure 1C) and numbers of MPO-positive cells (Figure 2) in Giardia GS/M-infected animals intracolonically instilled with 100 µg of TcdAB were not significantly different from TcdAB-administered controls.
Male C57BL/6 mice aged 4 to 6 weeks were infected with Giardia NF or GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdAB or PBS i.r. and, after 3 hours, animals were euthanized. Representative immunohistochemical images of MPO-positive cells (yellow) counterstained with DAPI (blue) in colonic tissues of uninfected animals or animals infected with Giardia NF or GS/M trophozoites following i.r. instillation of 100 µg TcdAB or PBS. All data are representative of two independent experiments (n = 5–11/group) and taken at the same magnification (400×). Bars equal 50 µm for all micrographs.
Giardia NF infections attenuate neutrophil-associated cytokines during TcdAB-induced colitis
A bead-based cytokine assay on colonic tissue samples was performed to determine whether Giardia NF infections attenuated protein levels of pro-inflammatory cytokines and chemokines associated with granulocyte tissue recruitment. Compared to control animals, intracolonic instillation of 100 µg of TcdAB significantly increased tissue levels of several PMN-associated mediators, including CXCL1, CXCL2, IL-17 and G-CSF (Figure 3). Colonic tissues collected from Giardia NF infected-animals administered TcdAB. demonstrated significantly lower protein levels of CXCL1, CXCL2, and IL-17 (Figure 3A to C), while G-CSF protein levels were similar from uninfected TcdAB-treated animals (Figure 3D). Notably, colonic protein levels of CXCL1, CXCL2, IL-17, and G-CSF in Giardia GS/M-infected animals were similar from uninfected TcdAB-treated animals (Figure 3E to H). These results support above observations that granulocyte infiltration is not attenuated in Giardia GS/M infected animals instilled with TcdAB, and suggest attenuation of granulocyte infiltration in Giardia NF-infected animals given TcdAB may result from decreased expression of PMN-associated mediators. These results also indicate that Giardia-mediated attenuation of PMN-associated mediators induced via TcdAB occurs in an isolate-dependent manner.
Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF or GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdAB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) CXCL1, (B) CXCL2, (C) IL-17, and (D) G-CSF were compared between Giardia NF-infected animals and uninfected animals given i.r. 100 µg of TcdAB or PBS. Levels of (E) CXCL1, (F) CXCL2, (G) IL-17, and (H) G-CSF were compared between Giardia GS/M-infected animals and uninfected animals given i.r. 100 µg of TcdAB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. n.s. = not significant * p<0.05.
Giardia GSM reduce colonic expression of several pro-inflammatory cytokines during TcdAB-induced colitis
After focusing on neutrophil-released cytokines, we investigated whether Giardia infections attenuated colonic expression of other pro-inflammatory cytokines. Compared to uninfected animals given PBS, intracolonic instillation of TcdAB to uninfected animals also resulted in heightened protein levels of CCL2, IL-6, and leukocyte inhibitory factor (LIF) (Figure 4) and IL-1β, IL-5, CXCL10, and CCL11 (Figure S1). Colonic protein levels of CCL2, IL-6, and LIF were significantly reduced in Giardia NF-infected animals given intracolonically with TcdAB (Figure 4A to C). In addition, colonic IL-12p70 levels were significantly greater in uninfected TcdAB-treated controls when compared to Giardia NF infected animals treated with TcdAB (Figure 4D). Conversely, CCL2, IL-6, LIF, and IL-12p70 levels in Giardia GS/M-infected animals treated with TcdAB were not significantly different from animals given TcdAB without giardiasis (Figure 4E to H). These results demonstrate that G. duodenalis NF attenuate colonic levels of CCL2, IL-6, LIF, and, potentially, IL-12p70 induced by 100 µg intracolonically of TcdAB in an isolate-dependent manner. Giardia NF-infections was unable to attenuate colonic expression levels of IL-1β, IL-5, CXCL10, and CCL11 (Fig. S1) Protein levels of IL-1α, IL-7, IL-9, IL-10, IL-12p40, IL-15, IFN-γ, CCL3, CCL4, CXCL9, VEGF remained unchanged between all animal groups (Figure S2). Therefore, following TcdAB instillation Giardia NF specifically targeted and decreased colonic expression of pro-inflammatory cytokines that recruit PMNs (CXCL1, CXCL2, and IL-17) and other cytokines (CCL2, IL-6, and LIF).
Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF or GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdAB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) CCL2, (B) IL-6, (C) LIF, and (D) IL-12p70 were compared between Giardia NF-infected animals and uninfected animals given i.r. 100 µg of TcdAB or PBS. Levels of (E) CCL2, (F) IL-6, (G) LIF, and (H) IL-12p70 were compared between Giardia GS/M-infected animals and uninfected animals given i.r. 100 µg of TcdAB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. n.s. = not significant * p<0.05.
Bead-based cytokine analysis of colonic tissues indicated Giardia GS/M infections upregulated colonic expression levels of a subset of pro-inflammatory mediators, following TcdAB. In uninfected animals, colonic administration of TcdAB significantly increased colonic expression levels of IL-1β and CXCL10 (Figure 5), and protein levels of these mediators were significantly increased in Giardia GS/M-infected animals compared to uninfected TcdAB-treated animals (Figure 5). Giardia GS/M-infected animals given TcdAB also demonstrated increased colonic protein levels of several inflammatory mediators not initially increased via colonic instillation of TcdAB (Figure S3). This did not appear to be a global increase in expression of inflammatory mediators, as CCL3 and CCL11 concentrations were similar between Giardia GS/M-infected animals given TcdAB and uninfected TcdAB controls (Figure S4). Several inflammatory mediators also remained unchanged between all experimental groups (Figure S5). These results suggest Giardia GS/M infections enhance expression of pro-inflammatory cytokines following colonic instillation of TcdAB. These data indicate that Giardia-infections can enhance or decrease the expression of inflammatory mediators following administration of a bacterial toxin, events which are isolate-specific.
Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdAB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) IL-1β and (B) CXCL10 were compared between Giardia GS/M-infected animals and uninfected animals given i.r. 100 µg of TcdAB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. * p<0.05.
Giardia NF trophozoites attenuate various inflammatory mediators from inflamed human mucosal biopsy tissues ex vivo
Our above results demonstrated in vivo that Giardia NF attenuated PMN accumulation and expression of pro-inflammatory cytokines following colonic administration of TcdA/TcdB. Therefore, we next assessed whether this same isolate was capable of attenuating similar inflammatory mediators from inflamed human colonic tissue. Using a previously published model [41], Giardia NF trophozoites were co-incubated ex vivo with colonic mucosal biopsy tissues collected the descending colon of patients with active CD, where supernatants and biopsy tissue homogenates were analyzed via a bead-based cytokine assay. Supernatants collected from co-incubation of Giardia NF trophozoites with inflamed biopsy tissues displayed significantly reduced supernatant levels of cytokines associated with granulocyte recruitment, including IL-8/CXCL8, growth-related oncogene (GRO) family proteins (CXCL1-3), IL-17A, G-CSF, and GM-CSF (Figure 6). Co-incubation of Giardia NF trophozoites with biopsy tissues also resulted in decreased IL-8/CXCL8 levels detected within biopsy tissue homogenates (Figure 7A), but not G-CSF and GM-CSF.
Human descending colon mucosal biopsy tissues obtained from areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Supernatant levels of (A) IL-8/CXCL8, (B) GRO, (C) CCL3, (D) IL-17A, (E) G-CSF, and (F) GM-CSF were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. * p<0.05.
Human descending colon mucosal biopsy tissues obtained from areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Tissue homogenate levels of (A) CXCL8, (B) GRO, (C) CCL3, (D) IL-17A, (E) G-CSF, and (F) GM-CSF were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. * p<0.05.
As in vivo Giardia NF attenuated colonic levels of CCL2, IL-6, LIF, and, potentially, IL-12p70 (Figure 4), we investigated whether live trophozoites were capable of attenuating protein expression of other chemokines. Supernatant levels of CCL2-5, CCL7, CCL11, CCL22, CXCL10, and CX3CL1 were significantly reduced in biopsy tissues co-incubated with Giardia NF trophozoites compared to control biopsy tissues (Figure 8). Tissue homogenate levels of these mediators were similar when biopsy tissues were incubated in the presence or absence of Giardia NF trophozoites (Figure 9). Therefore, attenuation of these mediators by G. duodenalis NF trophozoites appears to occur following their release into supernatants. These results also suggest that Giardia NF trophozoites are capable of attenuating CCL2 in different experimental models of inflammation. Compared to control biopsy supernatant, co-incubation of Giardia NF trophozoites with inflamed biopsy tissues ex vivo resulted in significant attenuation of IL-6, IL-7, IL-10, IL-12p70, IFN-α, soluble CD40 ligand (sCD40L) and vascular endothelial growth factor (VEGF) (Figure 10). Tissue homogenate levels of IL-1α, IL-6, and TNF-α were significantly reduced in biopsy tissue homogenates co-incubated with Giardia NF trophozoites, compared to biopsy tissue homogenates incubated in the absence of Giardia NF trophozoites (Figure 11). These results suggest that Giardia NF trophozoites attenuate secreted IL-7, IL-10, IL-12p70, IFN-α, sCD40L, and VEGF from inflamed mucosal biopsy tissues ex vivo, while also attenuating tissue levels of IL-1α, IL-6, and TNF-α. These results also suggest that Giardia infections are capable of attenuating IL-6 and IL-12p70 in during an in vivo infection and following incubation with human intestinal biopsy tissues ex vivo. Our data also indicated that Giardia NF trophozoites did not attenuate the expression of pro-inflammatory cytokines (Figure S6 and S7) or growth factors (Figure S8 and S9) within supernatants or biopsy tissue homogenates.
Human descending colon mucosal biopsy tissues obtained from areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Supernatant levels of (A) CCL2, (B) CCL4, (C) CCL5, (D) CCL7, (E) CCL11, (F) CCL22, (G) CXCL10, and (H) CX3CL1 were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. * p<0.05.
Human descending colon mucosal biopsy tissues obtained from areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Tissue homogenate levels of (A) CCL2, (B) CCL4, (C) CCL5, (D) CCL7, (E) CCL11, (F) CCL22, (G) CXCL10, and (H) CX3CL1 were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. * p<0.05.
Human descending colon mucosal biopsy tissues obtained from areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Supernatant levels of (A) IL-1α, (B) IL-6, (C) IL-7, (D) IL-10, (E) IL-12p70, (F) IFNα, (G) TNFα, (H) sCD40L, and (I) VEGF were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. * p<0.05.
Human descending colon mucosal biopsy tissues obtained from areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Tissue homogenate levels of (A) IL-1α, (B) IL-6, (C) IL-7, (D) IL-10, (E) IL-12p70, (F) IFNα, (G) TNFα, (H) sCD40L, and (I) VEGF were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. * p<0.05.
Discussion
Results from the present study demonstrate novel mechanisms through which certain Giardia infections were able to attenuate pro-inflammatory responses elicited by an inflammatory bacterial toxin in vivo, or from inflamed human intestinal tissues ex vivo. Attenuation of granulocyte infiltration in Giardia NF infected animals given TcdAB occurred concomitantly with reduced colonic expression of several inflammatory mediators, including those associated with granulocyte tissue recruitment. Several of these factors were also decreased following co-incubation of Giardia NF trophozoites and ex vivo inflamed human colonic mucosal biopsy tissues, and, therefore, support our in vivo observations that Giardia NF trophozoites are capable of attenuating factors associated with PMN recruitment. These results also reinforce previous observations that Giardia trophozoites are capable of attenuating IL-8/CXCL8 production from intestinal tissues in vitro and ex vivo [41]. Furthermore, our data indicate that Giardia trophozoites can attenuate the production of additional inflammatory mediators from intestinal tissues of differing origin, strengthening the notion that infection with specific Giardia isolates may modulate host immune responses in the gut. Interestingly Giardia GS/M-infected animals did not display reduced granulocyte infiltration nor decreased expression of inflammatory mediators. On the contrary, Giardia GS/M infections enhanced the expression of several factors initially upregulated following TcdAB administration and increased the expression of several factors not initially induced by TcdAB exposure. Therefore, our data suggest that Giardia infections attenuate intestinal pro-inflammatory responses in an isolate-dependent manner, and attenuation of such responses is associated with reduced tissue granulocyte infiltration. The results show that Giardia NF attenuate a variety of pro-inflammatory mediators when co-incubated with inflamed colonic mucosal biopsy tissues ex vivo, but the inability to decrease expression of all mediators examined suggests this process may be targeted towards certain inflammatory mediators. Moreover, observations that some inflammatory mediators are preferentially degraded within supernatants while others within tissues suggests that Giardia NF trophozoites possess multiple mechanisms capable of attenuating expression of inflammatory mediators in inflamed intestinal mucosal biopsy tissues.
Excessive PMN infiltration and activation contribute to several gastrointestinal inflammatory disorders, including the inflammatory bowel diseases (IBD) and C. difficile colitis [55]–[57]. The present findings indicate that Giardia NF infections in vivo attenuate PMN tissue recruitment induced by a pro-inflammatory bacterial toxin by reducing tissue expression of mediators that can contribute to the tissue recruitment of PMNs. Release of ELR+ chemokines, such as CXCL1, CXCL2, and IL-8/CXCL8, from a variety of intestinal tissue-resident cells promotes PMN accumulation and chemotaxis through intestinal tissues. In mice, these chemokines include CXCL1 and CXCL2, while in humans this also includes IL-8/CXCL8 [58]–[61]. Separately, enhanced expression of G-CSF promotes the release of PMNs into the bloodstream [31], [62], in a process that can be initiated via IL-17 production [32], [33]. Our results suggest that Giardia NF infections attenuate granulocyte infiltration into colonic tissues following colonic. TcdA/B by reducing colonic expression levels of CXCL1, CXCL2, and IL-17; these results are supported by observations that Giardia GS/M infections failed to attenuate colonic granulocyte infiltration and expression of PMN-associated mediators. In addition, Giardia NF parasites attenuated supernatant levels of IL-8/CXCL8, GRO-family proteins (CXCL1-3), IL-17, G-CSF, and GM-CSF when co-incubated with ex vivo descending colon mucosal biopsy tissues. Together, these data show for the first time that Giardia infections attenuate the release of a variety of mediators associated with PMN recruitment in different experimental models of inflammation, in an isolate-dependent manner.
Further research is needed to demonstrate whether the results from this study explain how Giardia infections can, in a strain-dependent manner, modulate the incidence of diarrheal disease [20], [22], and create an environment that is favourable for colonization by other gastrointestinal pathogens [18]. Indeed, the data suggest that Giardia may attenuate pro-inflammatory responses elicited by other co-infecting pathogens that can cause diarrheal disease, while other strains may enhance its development. Following their recruitment to tissues, PMNs are known to induce multiple pathophysiological events within intestinal tissues capable of causing diarrhea [28], [42]–[44]. Therefore, Giardia NF infections may protect against the development of diarrheal disease by attenuating PMN recruitment. Giardia GS/M infections did not enhance granulocyte infiltration following colonic of TcdAB; however, these infections resulted in enhanced expression of several pro-inflammatory mediators that could, potentially, enhance the development of diarrheal disease via other pathways. For example, IL-1β and IFNγ have been shown to modulate intestinal barrier dysfunction and ion secretion, and, resultantly, cause diarrhea [63]–[68]. As PMNs are essential to host defence against a variety of pathogens, more research is needed to determine whether attenuation of PMN recruitment by Giardia may also facilitate colonization by other pathogens. To date, research in vitro and in vivo has demonstrated differences in the pathogenesis of Giardia isolates, but has failed to clearly establish whether these differences are assemblage-specific. [10], [50], [54], [69], [70].
Data from this study also demonstrate that Giardia infections reduce the expression of pro-inflammatory mediators not associated with PMN recruitment, in an isolate-dependent manner. Giardia NF infections attenuated colonic expression of IL-6 and the related cytokine LIF induced following i.r. TcdAB, whereas Giardia GS/M did not. Moreover, co-incubation of Giardia NF trophozoites with inflamed human intestinal mucosal biopsy tissues ex vivo also resulted in attenuation of supernatant and tissue IL-6. The ability of Giardia sp. to modulate IL-6 during in vivo infection remains incompletely understood. These observations are consistent with previous studies that have shown that animals infected with Giardia muris display decreased IL-6 levels in jejunal tissues 5 days post-infection [71], while mice infected with Giardia GS/M display elevated IL-6 intestinal tissue levels [72]. The release of IL-6 and the related cytokine LIF can induce the expression of acute phase response proteins, including CRP [73], [74]. As Tanzanian children infected with Giardia were found to have lower serum CRP than their non-infected counterparts [22], future studies should examine whether Giardia infections can modulate the acute phase response in a strain-dependent manner, and establish whether the parasite-mediated attenuation of IL-6 expression is involved in this process.
The present findings illustrate that Giardia NF infections attenuate colonic expression of CCL2 and IL-12p70 in vivo, as well as in inflamed human intestinal mucosal biopsy tissues ex vivo. CCL2 is an important chemokine for monocytes and macrophages (reviewed in [75]). Conversely, co-incubation of Giardia trophozoites with in vitro Caco-2 monolayers results in increased mRNA expression of CCL2 [76]. Additional research is required in order to understand how Giardia infections may modulate monocyte/macrophage recruitment. IL-12p70 is composed of IL-12p35 and IL-12p40, and is the bioactive form responsible for inducing helper T1 (TH1) adaptive immune responses ([77]). Recent research has demonstrated that Giardia trophozoites products are capable of attenuating IL-12p70 expression from dendritic cells (DCs) stimulated with bacterial lipopolysaccharide [78]. In contrast other studies found that Giardia products may increase DC IL-12p70 expression [79], [80]. More research is required in order to determine how Giardia infections may modulate CCL2 and IL-12p70 expression in the context of intestinal inflammatory responses. We have recently shown that Giardia cathepsin B (catB) cysteine proteases degrade intestinal epithelial IL-8/CXCL8 and can attenuate PMN chemotaxis [41]. It remains to be determined whether Giardia catB proteases are involved in attenuating PMN accumulation in vivo and during human giardiasis. Genetic manipulation of the Giardia genome with Cre/loxP system has recently been demonstrated [81]. This would be extremely beneficial in determining the anti-inflammatory potential of Giardia catB proteases in vivo. Whether Giardia may exert immune-modulatory effects using other secretory-excretory products apart from Giardia catB proteases remains largely unknown. Giardia arginine deiminase has been shown to attenuate nitric oxide production from intestinal epithelial cells [82]–[84], to inhibit intestinal epithelial proliferation [85], to induce expression of IL-12p70 from DCs [80], and to inhibit T-cell proliferation [84], however these effects have yet to be characterized in vivo.
In conclusion, results from this study demonstrate that Giardia infections can attenuate granulocyte infiltration and expression of PMN chemokines in an isolate-dependent manner in an in vivo model of infectious colitis. Giardia trophozoites were also able to reduce the expression of a variety of inflammatory mediators released from inflamed colonic mucosal biopsy tissues from CD patients. These findings establish the strain-dependent anti-inflammatory properties of Giardia in the intestine.
Supporting Information
Figure S1.
Giardia NF infections in vivo do not attenuate colonic expression levels of IL-1β, IL-5, CXCL10, or CCL11 during C. difficile TcdA/TcdB-induced colitis. Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF trophozoites. 7 days post-infection, animals were given 100 µg of TcdA/TcdB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) IL-1β, (B) IL-5, (C) CXCL10, and (D) CCL11 were compared between Giardia NF-infected animals and uninfected animals given i.r. 100 µg of TcdA/TcdB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. n.s. = not significant * p<0.05.
https://doi.org/10.1371/journal.pone.0109087.s001
(TIF)
Figure S2.
Giardia NF infections in vivo do not attenuate colonic expression levels of inflammatory mediators not increased during C. difficile TcdA/TcdB-induced colitis. Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia NF trophozoites. 7 days post-infection, animals were given 100 µg of TcdA/TcdB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of IL-1α (A), IL-2 (B), IL-7 (C), (D) IL-9, (E) IL-10, (F) IL-12p40, (G) IL-15, (H) IFNγ, (I) CCL3, (J) CCL4, (K) CXCL9 and (L) VEGF were compared between Giardia NF-infected animals and uninfected animals given i.r. 100 µg of TcdA/TcdB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM.
https://doi.org/10.1371/journal.pone.0109087.s002
(TIF)
Figure S3.
Giardia GS/M infections in vivo upregulate colonic expression levels of several inflammatory mediators not increased during C. difficile TcdA/TcdB-induced colitis. Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdA/TcdB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) IL-2, (B) IL-5, (C) IL-15, (D) IL-17, (E) IFNγ, (F) CXCL9, (G) CCL4, and (H) VEGF were compared between Giardia GS/M-infected animals and uninfected animals given i.r. 100 µg of TcdA/TcdB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. * p<0.05.
https://doi.org/10.1371/journal.pone.0109087.s003
(TIF)
Figure S4.
Giardia GS/M infections in vivo do not modulate colonic expression levels of CCL3 and CCL11 during C. difficile TcdA/TcdB-induced colitis. Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdA/TcdB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) CCL3 and (B) CCL11 were compared between Giardia GS/M-infected animals and uninfected animals given i.r. 100 µg of TcdA/TcdB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM. * p<0.05.
https://doi.org/10.1371/journal.pone.0109087.s004
(TIF)
Figure S5.
Giardia GS/M infections in vivo do not attenuate colonic expression levels of inflammatory mediators not increased during C. difficile TcdA/TcdB-induced colitis. Male C57BL/6 mice, aged 4 to 6 weeks, were infected with Giardia GS/M trophozoites. 7 days post-infection, animals were given 100 µg of TcdA/TcdB or PBS i.r. and, after 3 hours, animals were euthanized. Levels of (A) IL-1α, (B) IL-7, (C) IL-9, (D) IL-10, (E) IL-12p40, and (F) TNFα were compared between Giardia GS/M-infected animals and uninfected animals given i.r. 100 µg of TcdA/TcdB or PBS. Values were determined via bead-based cytokine assay. All data are representative of two independent experiments (n = 5–11/group) and represented as mean ± SEM.
https://doi.org/10.1371/journal.pone.0109087.s005
(TIF)
Figure S6.
Giardia NF trophozoites do attenuate supernatant levels of several inflammatory mediators released from descending colon mucosal biopsy tissues. Human descending colon mucosal biopsy tissues obtained in areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Supernatant levels of (A) IL-1ra, (B) IL-1β, (C) IL-2, (D) IL-12p40, (E) IL-13, (F) IL-15, (G) IFNγ, and (H) TNFβ were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. n.s. = not significant.
https://doi.org/10.1371/journal.pone.0109087.s006
(TIF)
Figure S7.
Giardia NF trophozoites do attenuate levels of several inflammatory mediators released from descending colon mucosal biopsy homogenates. Human descending colon mucosal biopsy tissues obtained in areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Tissue homogenate levels of (A) IL-1ra, (B) IL-1β, (C) IL-2, (D) IL-12p40, (E) IL-13, (F) IL-15, (G) IFNγ, and (H) TNFβ were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. n.s. = not significant.
https://doi.org/10.1371/journal.pone.0109087.s007
(TIF)
Figure S8.
Giardia NF trophozoites do attenuate supernatant levels of several growth factors released from descending colon mucosal biopsy tissues. Human descending colon mucosal biopsy tissues obtained in areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 Giardia NF trophozoites for 6 hours. Supernatant levels of (A) FGF, (B) Flt-3L, (C) PDGF-AA, (D) PDGF-BB, and (E) TGFβ were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. n.s. = not significant.
https://doi.org/10.1371/journal.pone.0109087.s008
(TIF)
Figure S9.
Giarida NF trophozoites do attenuate levels of several growth factors released from descending colon mucosal biopsy tissue homogenates. Human descending colon mucosal biopsy tissues obtained in areas of active inflammation from patients with active Crohn’s disease (CD) were incubated with 5.0×106 G. duodenalis NF trophozoites for 6 hours. Tissue homogenate levels of (A) FGF, (B) Flt-3L, (C) PDGF-AA, (D) PDGF-BB, and (E) TGFβ were determined via a bead-based cytokine assay. All data are representative of three independent experiments (n = 4–6/group) and represented as median with the range. n.s. = not significant.
https://doi.org/10.1371/journal.pone.0109087.s009
(TIF)
Author Contributions
Conceived and designed the experiments: JAC SAH AGB. Performed the experiments: JAC JPM. Analyzed the data: JAC AGB. Contributed reagents/materials/analysis tools: PLB AGB LPS. Contributed to the writing of the manuscript: JAC AGB.
References
- 1. Monis PT, Andrews RH, Mayrhofer G, Ey PL (1999) Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol 16: 1135–1144.
- 2. Lasek-Nesselquist E, Welch DM, Sogin ML (2010) The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol 40: 1063–1074.
- 3. Franzen O, Jerlstrom-Hultqvist J, Castro E, Sherwood E, Ankarklev J, et al. (2009) Draft genome sequencing of giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathog 5: e1000560.
- 4. Jerlstrom-Hultqvist J, Ankarklev J, Svard SG (2010) Is human giardiasis caused by two different Giardia species? Gut Microbes 1: 379–382.
- 5. Cotton JA, Beatty JK, Buret AG (2011) Host parasite interactions and pathophysiology in Giardia infections. Int J Parasitol 41: 925–933.
- 6. Wensaas KA, Langeland N, Hanevik K, Morch K, Eide GE, et al. (2012) Irritable bowel syndrome and chronic fatigue 3 years after acute giardiasis: historic cohort study. Gut 61: 214–219.
- 7. Halliez MC, Buret AG (2013) Extra-intestinal and long term consequences of Giardia duodenalis infections. World J Gastroenterol 19: 8974–8985.
- 8. Oberhuber G, Kastner N, Stolte M (1997) Giardiasis: a histologic analysis of 567 cases. Scand J Gastroenterol 32: 48–51.
- 9. Scott KG, Meddings JB, Kirk DR, Lees-Miller SP, Buret AG (2002) Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 123: 1179–1190.
- 10. Chin AC, Teoh DA, Scott KG, Meddings JB, Macnaughton WK, et al. (2002) Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase-3-dependent manner. Infect Immun 70: 3673–3680.
- 11. Troeger H, Epple HJ, Schneider T, Wahnschaffe U, Ullrich R, et al. (2007) Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum. Gut 56: 328–335.
- 12. Hanevik K, Hausken T, Morken MH, Strand EA, Morch K, et al. (2007) Persisting symptoms and duodenal inflammation related to Giardia duodenalis infection. J Infect 55: 524–530.
- 13. Chen TL, Chen S, Wu HW, Lee TC, Lu YZ, et al. (2013) Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice. Gut Pathog 5: 26.
- 14. Ankarklev J, Jerlstrom-Hultqvist J, Ringqvist E, Troell K, Svard SG (2010) Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 8: 413–422.
- 15. Hagel I, Cabrera M, Puccio F, Santaella C, Buvat E, et al. (2011) Co-infection with Ascaris lumbricoides modulates protective immune responses against Giardia duodenalis in school Venezuelan rural children. Acta Trop 117: 189–195.
- 16. Wang L, Xiao L, Duan L, Ye J, Guo Y, et al. (2013) Concurrent infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS Negl Trop Dis 7: e2437.
- 17. Ankarklev J, Hestvik E, Lebbad M, Lindh J, Kaddu-Mulindwa DH, et al. (2012) Common coinfections of Giardia intestinalis and Helicobacter pylori in non-symptomatic Ugandan children. PLoS Negl Trop Dis 6: e1780.
- 18. Mukherjee AK, Chowdhury P, Rajendran K, Nozaki T, Ganguly S (2014) Association between Giardia duodenalis and Coinfection with Other Diarrhea-Causing Pathogens in India. Biomed Res Int 2014: 786480.
- 19. Oberhelman RA, Flores-Abuxapqui J, Suarez-Hoil G, Puc-Franco M, Heredia-Navarrete M, et al. (2001) Asymptomatic salmonellosis among children in day-care centers in Merida, Yucatan, Mexico. Pediatr Infect Dis J 20: 792–797.
- 20. Bilenko N, Levy A, Dagan R, Deckelbaum RJ, El-On Y, et al. (2004) Does co-infection with Giardia lamblia modulate the clinical characteristics of enteric infections in young children? Eur J Epidemiol 19: 877–883.
- 21. Bhavnani D, Goldstick JE, Cevallos W, Trueba G, Eisenberg JN (2012) Synergistic effects between rotavirus and coinfecting pathogens on diarrheal disease: evidence from a community-based study in northwestern Ecuador. Am J Epidemiol 176: 387–395.
- 22. Veenemans J, Mank T, Ottenhof M, Baidjoe A, Mbugi EV, et al. (2011) Protection against diarrhea associated with Giardia intestinalis Is lost with multi-nutrient supplementation: a study in Tanzanian children. PLoS Negl Trop Dis 5: e1158.
- 23. Kostman R (1975) Infantile genetic agranulocytosis. A review with presentation of ten new cases. Acta Paediatr Scand 64: 362–368.
- 24. Zeidler C, Germeshausen M, Klein C, Welte K (2009) Clinical implications of ELA2-, HAX1-, and G-CSF-receptor (CSF3R) mutations in severe congenital neutropenia. Br J Haematol 144: 459–467.
- 25. Kuijpers T, Lutter R (2012) Inflammation and repeated infections in CGD: two sides of a coin. Cell Mol Life Sci 69: 7–15.
- 26. Szabady RL, McCormick BA (2013) Control of neutrophil inflammation at mucosal surfaces by secreted epithelial products. Front Immunol 4: 220.
- 27. Fournier BM, Parkos CA (2012) The role of neutrophils during intestinal inflammation. Mucosal Immunol 5: 354–366.
- 28. Chin AC, Parkos CA (2007) Pathobiology of neutrophil transepithelial migration: implications in mediating epithelial injury. Annu Rev Pathol 2: 111–143.
- 29. Summers C, Rankin SM, Condliffe AM, Singh N, Peters AM, et al. (2010) Neutrophil kinetics in health and disease. Trends Immunol 31: 318–324.
- 30. Kolaczkowska E, Kubes P (2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13: 159–175.
- 31. Wengner AM, Pitchford SC, Furze RC, Rankin SM (2008) The coordinated action of G-CSF and ELR+CXC chemokines in neutrophil mobilization during acute inflammation. Blood 111: 42–49.
- 32. Mei J, Liu Y, Dai N, Hoffmann C, Hudock KM, et al. (2012) Cxcr2 and Cxcl5 regulate the IL-17/G-CSF axis and neutrophil homeostasis in mice. J Clin Invest 122: 974–986.
- 33. Schwarzenberger P, Huang W, Ye P, Oliver P, Manuel M, et al. (2000) Requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17-mediated granulopoiesis. J Immunol 164: 4783–4789.
- 34. Semerad CL, Liu F, Gregory AD, Stumpf K, Link DC (2002) G-CSF is an essential regulator of neutrophil trafficking from the bone marrow to the blood. Immunity 17: 413–423.
- 35. Massena S, Christoffersson G, Hjertstrom E, Zcharia E, Vlodavsky I, et al. (2010) A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils. Blood 116: 1924–1931.
- 36. Ley K, Laudanna C, Cybulsky MI, Nourshargh S (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7: 678–689.
- 37. Chou RC, Kim ND, Sadik CD, Seung E, Lan Y, et al. (2010) Lipid-cytokine-chemokine cascade drives neutrophil recruitment in a murine model of inflammatory arthritis. Immunity 33: 266–278.
- 38. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, et al. (2010) Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330: 362–366.
- 39. Lee HY, Hyung S, Lee NY, Yong TS, Han SH, et al. (2012) Excretory-secretory products of Giardia lamblia induce interleukin-8 production in human colonic cells via activation of p38, ERK1/2, NF-kappaB and AP-1. Parasite Immunol 34: 183–198.
- 40. Fisher BS, Estrano CE, Cole JA (2013) Modeling long-term host cell-Giardia lamblia interactions in an in vitro co-culture system. PLoS One 8: e81104.
- 41.
Cotton JA, Bhargava A, Ferraz JG, Yates RM, Beck PL, et al. (2014) Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis. Infect Immun.
- 42. Madara JL, Patapoff TW, Gillece-Castro B, Colgan SP, Parkos CA, et al. (1993) 5′-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J Clin Invest 91: 2320–2325.
- 43. Strohmeier GR, Lencer WI, Patapoff TW, Thompson LF, Carlson SL, et al. (1997) Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia. J Clin Invest 99: 2588–2601.
- 44. Weissmuller T, Campbell EL, Rosenberger P, Scully M, Beck PL, et al. (2008) PMNs facilitate translocation of platelets across human and mouse epithelium and together alter fluid homeostasis via epithelial cell-expressed ecto-NTPDases. J Clin Invest 118: 3682–3692.
- 45.
Hirota SA, Fines K, Ng J, Traboulsi D, Lee J, et al. (2010) Hypoxia-inducible factor signaling provides protection in Clostridium difficile-induced intestinal injury. Gastroenterology 139: 259–269 e253.
- 46. Teoh DA, Kamieniecki D, Pang G, Buret AG (2000) Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance. J Parasitol 86: 800–806.
- 47. Aggarwal A, Merritt JW Jr, Nash TE (1989) Cysteine-rich variant surface proteins of Giardia lamblia. Mol Biochem Parasitol 32: 39–47.
- 48. Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72: 431–432.
- 49. Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77: 487–488.
- 50. Solaymani-Mohammadi S, Singer SM (2011) Host immunity and pathogen strain contribute to intestinal disaccharidase impairment following gut infection. J Immunol 187: 3769–3775.
- 51. Singer SM, Nash TE (2000) The role of normal flora in Giardia lamblia infections in mice. J Infect Dis 181: 1510–1512.
- 52. Hirota SA, Iablokov V, Tulk SE, Schenck LP, Becker H, et al. (2012) Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure. Infect Immun 80: 4474–4484.
- 53. Arndt H, Kubes P, Grisham MB, Gonzalez E, Granger DN (1992) Granulocyte turnover in the feline intestine. Inflammation 16: 549–559.
- 54. Bartelt LA, Roche J, Kolling G, Bolick D, Noronha F, et al. (2013) Persistent G. lamblia impairs growth in a murine malnutrition model. J Clin Invest 123: 2672–2684.
- 55. Kumar NB, Nostrant TT, Appelman HD (1982) The histopathologic spectrum of acute self-limited colitis (acute infectious-type colitis). Am J Surg Pathol 6: 523–529.
- 56. Chin AC, Parkos CA (2006) Neutrophil transepithelial migration and epithelial barrier function in IBD: potential targets for inhibiting neutrophil trafficking. Ann N Y Acad Sci 1072: 276–287.
- 57. Kelly CP, Becker S, Linevsky JK, Joshi MA, O'Keane JC, et al. (1994) Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit. J Clin Invest 93: 1257–1265.
- 58. Spehlmann ME, Dann SM, Hruz P, Hanson E, McCole DF, et al. (2009) CXCR2-dependent mucosal neutrophil influx protects against colitis-associated diarrhea caused by an attaching/effacing lesion-forming bacterial pathogen. J Immunol 183: 3332–3343.
- 59. Ranganathan P, Jayakumar C, Manicassamy S, Ramesh G (2013) CXCR2 knockout mice are protected against DSS-colitis-induced acute kidney injury and inflammation. Am J Physiol Renal Physiol 305: F1422–1427.
- 60. Jamieson T, Clarke M, Steele CW, Samuel MS, Neumann J, et al. (2012) Inhibition of CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis. J Clin Invest 122: 3127–3144.
- 61. Buanne P, Di Carlo E, Caputi L, Brandolini L, Mosca M, et al. (2007) Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice. J Leukoc Biol 82: 1239–1246.
- 62. Eash KJ, Greenbaum AM, Gopalan PK, Link DC (2010) CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow. J Clin Invest 120: 2423–2431.
- 63. Utech M, Ivanov AI, Samarin SN, Bruewer M, Turner JR, et al. (2005) Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane. Mol Biol Cell 16: 5040–5052.
- 64. Wang F, Schwarz BT, Graham WV, Wang Y, Su L, et al. (2006) IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology 131: 1153–1163.
- 65. Al-Sadi R, Guo S, Ye D, Dokladny K, Alhmoud T, et al. (2013) Mechanism of IL-1beta modulation of intestinal epithelial barrier involves p38 kinase and activating transcription factor-2 activation. J Immunol 190: 6596–6606.
- 66. Paul G, Marchelletta RR, McCole DF, Barrett KE (2012) Interferon-gamma alters downstream signaling originating from epidermal growth factor receptor in intestinal epithelial cells: functional consequences for ion transport. J Biol Chem 287: 2144–2155.
- 67. Sugi K, Musch MW, Field M, Chang EB (2001) Inhibition of Na+, K+-ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function. Gastroenterology 120: 1393–1403.
- 68. Bertelsen LS, Eckmann L, Barrett KE (2004) Prolonged interferon-gamma exposure decreases ion transport, NKCC1, and Na+-K+-ATPase expression in human intestinal xenografts in vivo. Am J Physiol Gastrointest Liver Physiol 286: G157–165.
- 69. Panaro MA, Cianciulli A, Mitolo V, Mitolo CI, Acquafredda A, et al. (2007) Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis. FEMS Immunol Med Microbiol 51: 302–309.
- 70. Cevallos A, Carnaby S, James M, Farthing JG (1995) Small intestinal injury in a neonatal rat model of giardiasis is strain dependent. Gastroenterology 109: 766–773.
- 71. Scott KG, Logan MR, Klammer GM, Teoh DA, Buret AG (2000) Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Infect Immun 68: 3412–3418.
- 72. Zhou P, Li E, Zhu N, Robertson J, Nash T, et al. (2003) Role of interleukin-6 in the control of acute and chronic Giardia lamblia infections in mice. Infect Immun 71: 1566–1568.
- 73. Pepys MB, Hirschfield GM (2003) C-reactive protein: a critical update. J Clin Invest 111: 1805–1812.
- 74. Mayer P, Geissler K, Ward M, Metcalf D (1993) Recombinant human leukemia inhibitory factor induces acute phase proteins and raises the blood platelet counts in nonhuman primates. Blood 81: 3226–3233.
- 75. Deshmane SL, Kremlev S, Amini S, Sawaya BE (2009) Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 29: 313–326.
- 76. Roxstrom-Lindquist K, Ringqvist E, Palm D, Svard S (2005) Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect Immun 73: 8204–8208.
- 77. Gee K, Guzzo C, Che Mat NF, Ma W, Kumar A (2009) The IL-12 family of cytokines in infection, inflammation and autoimmune disorders. Inflamm Allergy Drug Targets 8: 40–52.
- 78. Kamda JD, Singer SM (2009) Phosphoinositide 3-kinase-dependent inhibition of dendritic cell interleukin-12 production by Giardia lamblia. Infect Immun 77: 685–693.
- 79. Banik S, Renner Viveros P, Seeber F, Klotz C, Ignatius R, et al. (2013) Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia. Infect Immun 81: 2309–2317.
- 80. Obendorf J, Renner Viveros P, Fehlings M, Klotz C, Aebischer T, et al. (2013) Increased expression of CD25, CD83, and CD86, and secretion of IL-12, IL-23, and IL-10 by human dendritic cells incubated in the presence of Toll-like receptor 2 ligands and Giardia duodenalis. Parasit Vectors 6: 317.
- 81. Wampfler PB, Faso C, Hehl AB (2014) The Cre/loxP system in Giardia lamblia: genetic manipulations in a binucleate tetraploid protozoan. Int J Parasitol 44: 497–506.
- 82. Ringqvist E, Palm JE, Skarin H, Hehl AB, Weiland M, et al. (2008) Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol Biochem Parasitol 159: 85–91.
- 83. Eckmann L, Laurent F, Langford TD, Hetsko ML, Smith JR, et al. (2000) Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia. J Immunol 164: 1478–1487.
- 84. Stadelmann B, Hanevik K, Andersson MK, Bruserud O, Svard SG (2013) The role of arginine and arginine-metabolizing enzymes during Giardia - host cell interactions in vitro. BMC Microbiol 13: 256.
- 85. Stadelmann B, Merino MC, Persson L, Svard SG (2012) Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells. PLoS One 7: e45325.