Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate

Abstract

Giardia intestinalis is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major Giardia assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10 h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., cxcl1-3, ccl2, 1l1a, and il1b) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A Giardia, we identified the induction of novel cytokines (cxcl8, cxcl10, csf1, cx3cl1, il12a, il11) and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NFκB) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during Giardia assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages.

The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites

“…Giardiasis is also a problem in domestic animals, and the zoonotic potential of Giardia has been highlighted in recent years [5]. In vitro models of the interaction of G. intestinalis with human cells have helped to unravel clues to how Giardia causes disease [6–8], such as the importance of, the adhesive disc for attachment [9], flagella for motility [10, 11], secreted cysteine proteases for interference with host defenses [12–16], interactions with the intestinal microbiota [6, 17], differentiation into cysts for transmission [4, 18] and interference with nitric oxide (NO) production [19, 20]. Despite this progress it remains uncertain whether these in vitro models are representative of the natural infection, particularly because animal models of G. intestinalis infection have significant limitations.…”

The Chymase Mouse Mast Cell Protease-4 Regulates Intestinal Cytokine Expression in Mature Adult Mice Infected with Giardia intestinalis

“…Accumulated data suggest that there is a mixed Th1/Th2/Th17 response during giardiasis [19,20]. When G. intestinalis attach to the microvillus brush border of intestinal epithelial cells (IECs) there is a production of chemokines and cytokines that will attract immune cells to the intestinal submucosa [20][21][22]. However, the effects differ depending on model systems used.…”

“…However, the effects differ depending on model systems used. In cultured human IECs challenged by G. intestinalis trophozoites (assemblage B, isolate GS), several chemokines were highly up-regulated early-at 1.5 h after challenge [21]. In experimental infections of gerbils with the WB isolate (ATCC 50803) several chemokines and cytokines was up-regulated [20], whereas no major up-regulation of chemokine or cytokine genes were seen in 5-6-week-old female mice infected with trophozoites of the GS isolate [22].…”

Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

“… 27 Importantly, these stressors have the potential to significantly increase with a change in diet or gastrointestinal infection. 28 , 29 , 30 Discovering proteins critical to the epithelial response to oxidative insult has the potential to identify therapeutic targets to prevent tissue damage that ultimately results in a leaky gut phenotype. This occurs after the breakdown of the epithelial barrier of the gastrointestinal tract, increasing translocation of luminal antigens, microbes, and their products, which leads to increased local and systemic inflammation.…”