Investigation of Host-Parasite Interaction in Giardia muris infection via a fluorescent Ca²⁺ reporter - (POP-WS)

Abstract text

Introduction

Infections with the protozoan parasite Giardia duodenalis cause a global health burden with over 200 million symptomatic cases every year. Giardia muris, a natural parasite in mice, serves as a model for the asymptomatic course seen in many cases of G. duodenalis infection. Our previous work suggests that modest adaptive immune responses operate together with the intestinal accumulation of pro-inflammatory myeloid cells, allowing for the control of infection in absence of overt immunopathology. Whether attachment of Giardia trophozoites directly drives immune defense by cells forming the intestinal epithelial barrier is not clear.

Objectives

As macrophages develop a pro-inflammatory phenotype upon activation of the mechano-sensitive Ca²⁺ channel PIEZO-1, we address whether attachment of G. muris to myeloid cells results in mechano-sensing driven defense mechanisms at the site of infection.

Materials & Methods

Using 2-Photon microscopy, Bone Marrow Derived Macrophages (BMDM) from LysM-GCaMP6f reporter mice allow the visualization of PIEZO-1 induced Ca²⁺ influx based on fluorescence intensity. The performance of the system was tested by exposure of BMDM to Ca²⁺ ionophores, PIEZO-1 agonists and by direct mechanical stimulation applied by nanoindentation via Atomic Force Microscopy (AFM). Finally, BMDM were exposed to live G. muris trophozoites.

Results

Stimulation of BMDM with ionomycin caused an immediate increase in intracellular Ca²⁺, which was blocked by the addition of Ca²⁺ chelators. The application of the PIEZO-1 specific agonist YODA caused a similar effect, which could be reverted by the application of YODA antagonist GsMTx4. Furthermore, mechanostimulation of BMDM by nanoindentation via Atomic Force Microscopy initiated an increase in Ca²⁺ signal in the stimulated cells. Importantly, this finding was mirrored by a clear increase in Ca²⁺ reporter signal intensity in BMDM exposed to live G. muris trophozoites.

Conclusions

The LysM-GCaMP6f system reports the activation of mechanosensitive Ca²⁺ channels in myeoloid cells. Whether the attachment process of Giardia trophozoites results in the PIEZO-1 dependent activation of myeoloid cells and thereby triggers a local defense program against G. muris infection remains to be investigated.