PLoS One. 2014. Oct 7:9(10):e109087

Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

Cotton JA, Motta JP, Schenck LP, Hirota SA, Beck PL, Buret AG

Department of Biological Sciences, University of Calgary, 2500 University Dr NW, Calgary, AB, Canada, T2N 1N4

 

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 paralleled 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.

PMID: 25289678

 

Supplement

Giardia duodenalis (syn. G. intestinalis, G. lamblia) infections are a leading cause of waterborne diarrheal disease and included on the WHO’s Neglected Disease Initiative. The parasite is divided into eight distinct genetic assemblages designated as “A” through “H”, but it has been suggested that assemblage A and B are two separate Giardia species. At peak infection, motile trophozoite numbers exceeds 106 trophozoites per centimetre of gut and induce pathophysiological processes culminating in malabsorptive diarrheal disease. Despite this, the intestinal mucosa of the majority of Giardia-infected individuals is devoid of overt signs of inflammation.

Previous reports have suggested Giardia infections modulate their host’s inflammatory responses. Tanzanian children infected with Giardia had reduced incidences of diarrheal disease, fever, and lower serum C-reactive protein compared to children not infected with Giardia [1]. Our research focuses on molecular mechanisms via which G. duodenalis trophozoites modulate host pro-inflammatory responses. We previously demonstrated G. duodenalis trophozoites release cathepsin B cysteine proteases that degrade the potent neutrophil (PMN) chemokine interleukin-8 (CXCL8) and attenuate CXCL8-induced PMN chemotaxis [2]. It remained unknown whether Giardia infections could attenuate pro-inflammatory responses in an in vivo model of bacterial-toxin-induced inflammation and whether parasites were capable of down regulating pro-inflammatory responses in chronically inflamed intestinal tissues. We hypothesized in vivo G. duodenalis infections could attenuate acute intestinal pro-inflammatory responses induced by a potent bacterial toxin and that trophozoites modulate pro-inflammatory signals released from chronically inflamed intestinal tissues.

Our data partially supported this hypothesis and was published in PLoS One in 2014 [3]. Results demonstrated assemblage A G. duodenalis NF infections attenuated granulocyte infiltration induced via intra-rectal (i.r.) instillation of Clostridium difficile toxin A/B (TcdA/B). Animals infected with the G. duodenalis assemblage B GS/M isolate did not display significantly reduced granulocyte infiltration compared to uninfected controls administered TcdA/B. This method of acute intestinal inflammation has been described previously [4].

Colonic expression of the PMN-associated cytokines and chemokines CXCL1, CXCL2, and interleukin(IL)-17 in animals infected with G. duodenalis NF and administered TcdA/B i.r. was significantly reduced compared to uninfected controls. Supporting above results, G. duodenalis GS/M-infected animals did not display reduced expression of these PMN-associated cytokines/chemokines.

Colonic tissues were analyzed to determine whether G. duodenalis infections modulated expression of other inflammatory mediators. Compared to uninfected animals, colonic tissues from G. duodenalis NF-infected animals displayed attenuated tissue levels of CCL2, IL-6, and the IL-6-related cytokine leukemia inhibitor factor (LIF) following i.r. TcdA/B. These mediators were not reduced in in vivo G. duodenalis GS/M infections administered TcdA/B. When compared against uninfected controls, G. duodenalis GS/M-infected animals administered TcdA/B displayed elevated levels of IL-1β and CXCL10.

Finally, we analyzed whether G. duodenalis NF trophozoites could attenuate pro-inflammatory signals released from mucosal biopsy tissues collected from the descending colon of patients with Crohn’s disease. This procedure has been described previously [2, 5]. Mucosal biopsy tissues co-incubated with G. duodenalis NF trophozoites displayed decreased supernatant levels of PMN-associated cytokines/chemokines, including CXCL8, growth related oncogenes (GROs), granulocyte colony stimulating factor (G-CSF), and granulocyte/monocyte colony stimulating factor (GM-CSF). In addition, parasites decreased supernatant levels of several other chemokines and cytokines.

This study indicates certain G. duodenalis infections are capable of attenuating acute intestinal inflammatory responses in an in vivo model of acute intestinal inflammation and chronically inflamed gastrointestinal tissues. These findings are summarized in Figure 1. As Giardia infections have been observed in association with a variety of other gastrointestinal pathogens, it’s possible that the immunomodulatory mechanisms of the parasite may, potentially, attenuate intestinal inflammatory responses to co-infecting pathogens.

 

ab fig1-1

 

References

  1. Veenemans J, Mank T, Ottenhof M, Baidjoe A, Mbugi EV, Demir AY, et al. Protection against diarrhea associated with Giardia intestinalis Is lost with multi-nutrient supplementation: a study in Tanzanian children. PLoS Negl Trop Dis. 2011;5(6):e1158. doi: 10.1371/journal.pntd.0001158. PubMed PMID: 21666789; PubMed Central PMCID: PMC3110167.
  2. Cotton JA, Bhargava A, Ferraz JG, Yates RM, Beck PL, Buret AG. Giardia duodenalis cathepsin B proteases degrade intestinal epithelial interleukin-8 and attenuate interleukin-8-induced neutrophil chemotaxis. Infect Immun. 2014;82(7):2772-87. doi: 10.1128/IAI.01771-14. PubMed PMID: 24733096; PubMed Central PMCID: PMC4097641.
  3. Cotton JA, Motta JP, Schenck LP, Hirota SA, Beck PL, Buret AG. 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. 2014;9(10):e109087. doi: 10.1371/journal.pone.0109087. PubMed PMID: 25289678; PubMed Central PMCID: PMC4188619.
  4. Hirota SA, Iablokov V, Tulk SE, Schenck LP, Becker H, Nguyen J, et al. 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. 2012;80(12):4474-84. doi: 10.1128/IAI.00933-12. PubMed PMID: 23045481; PubMed Central PMCID: PMC3497439.
  5. Ng J, Hirota SA, Gross O, Li Y, Ulke-Lemee A, Potentier MS, et al. Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome. Gastroenterology. 2010;139(2):542-52, 52 e1-3. doi: 10.1053/j.gastro.2010.04.005. PubMed PMID: 20398664.

 

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