Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Host-microbiota interactions

• Epithelial cell biology

• Type 2 immunity

Main Findings

In this preprint, the authors investigate the hypothesis that intestinal epithelial Tuft cells (TC) actively participate in gut barrier defence against enteric helminths. Based on work previously published, the authors speculate that TC serve as source of Acetylcholine (ACh) to support type 2 immunity. To investigate if TC contribute to the production of ACh in the gut they investigate Choline-Acetyl-Transferase (ChAT) expression in epithelial organoids generated from Pou2f3+/+and Pou2f3-/-(TC-deficient) crypts. The authors confirm the absence of TC, which coincides with the absence of ChAT expression in Pou2f3-deficient organoids. Using a combination of immunofluorescence and in situ hybridization the authors confirm that ChAT mRNAs are exclusively found in Dclk1-expressing TC in the epithelium. Using a helminth infection model (H.poly) the authors next identify that parasitic infections increase intestinal TC numbers but doesn’t affect ChAT expression. Employing an inducible and conditional deletion system for ChAT in intestinal epithelial cells (VillinCreERT2xChatloxP/loxP) the authors then address the role of epithelial cell-derived ACh on parasite burden and fecal egg release. Demonstrating the absence of ACh within intestinal TC following the induction of Cre-recombinase activity, the authors report elevated levels of adult worms in the intestines of ChAT-deficient mice, as well as an increased release of eggs if TC are unable to produce ACh. To determine if the lack in controlling the parasite is a result of an altered activation of immune cells, or an impairment in Goblet cells-mediated anti-microbial activity, the researchers quantify Gata3+ immune cells, Goblet cells and Retnlbexpression. Absence of ChAT in epithelial cells did not impact the ability of the host to mount an appropriate activation of the immune system, but instead suggested that TC-derived ACh act downstream of immune cell activation. Quantifying the levels of luminal ACh in helminth infected mice revealed an increase in luminal ACh. Absence of luminal ACh in VillinCreERT2xChatloxP/loxPmice revealed a greater metabolic fitness of the parasite. These observations suggest that TC-derived ACh directly impact the helminth by acting on parasite-expressed muscarinic or nicotinic ACh receptors. Using in vitro cultures of H.poly, the authors demonstrate that ACh, similar to the widely used anti-parasitic ACh receptor (AChR) agonist Levamisole, impairs the fertility of the parasite and its ability to produce eggs. Addressing the nature of the AChR through selective inhibition of either muscarinic or nicotinic AChR, the authors demonstrate that ACh-mediated impairment of parasitic fertility operates through muscarinic AChR expressed by the parasite. Their findings collectively demonstrate that intestinal TC are a prominent source of ACh which actively participates in the defence against enteric helminths by altering the the parasite’s fertility via muscarinic AChR.

Limitations

• The authors demonstrate elevated Retnlb levels and ACh levels in the gut. A better discussion on how both molecules impact parasitic fitness would be needed. Are Retnlb or ACh directly affecting ATP levels within the worm?

• It remains unclear if AChR activity on worms is needed for colonization and infection. A more detailed assessment of worm numbers and L4 larvae counts early and later during infection would be supportive data.

• One wonders if ChAT expression in TC is driven by IL-13 triggered STAT6 activation even in the steady state.

• It would be interesting to see if luminal application of ACh restores the impaired anti-parasitic effects in VillinCreERT2xChatloxP/loxPmice in vivo.

• Even though likely difficult to achieve, a more thorough quantification of immune cell functions (IL-13, IL-5, AREG production) and immune cells numbers would be helpful to strength the authors data separating TC-derived immune modulation from TC-derived anti-parasitic activity.

• Is ChAT expression in TC anatomically restricted?

• Is the expression Acetylcholine-Esterase within the H.poly regulated or a constitutive feature?

Significance/Novelty

What is the novelty of the preprint for the field specific?

The authors provide strong evidence that TC are an active part of the anti-parasitic defence arsenal and therefore extend their accepted role as parasitic sentinels in the gut. The provided data raises several intriguing questions on the directionality of vesicle release by TC (luminal ACh), the role of epithelial neurotransmitters in the modulation of enteric parasites and potential mechanisms of resistance by the parasite.

How does the result of the preprint matter for general immunologists and/or patients?

This article nicely demonstrates that ACh production by intestinal TC directly aids in anti-parasitic defence through direct modulation of the parasite.

Credit

Reviewed by Arthur Mortha (University of Toronto, Department of Immunology) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interest in relation to their involvement in the review.