Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Mucosal Immunology

• Tuft cell – ILC2 crosstalk

• Anti-helminth immunity

Main Findings

In this preprint, the authors explored the intricate interactions between tuft cells and type 2 innate lymphoid cells (ILC2s) within the intestine. They aimed to understand how tuft cells regulate immune responses through their production of IL-25, particularly in response to microbe-derived signals and helminth infections. Using conditional knockout mice to target the IL-25 receptor (IL-17RB; Il17rbfl/fl) in ILC2s (e.g., Nmur1-iCre-eGFP) or tuft cells (e.g., Il25-iCre), the researchers demonstrated the essential role of ILC2 intrinsic IL-17RB in the activation of ILC2s by IL-25 and showed that tuft cell intrinsic IL-17RB restrains IL-25 bioavailability, preventing tonic ILC2 stimulation. This validated and built upon previous knowledge of the tuft cell-ILC2 circuit. They then employed transgenic mouse lines that genetically encode the calcium indicator GCaMP6 in tuft cells (Trpm5-cre; R26-GCaMP6f) or were deficient in the calcium channel inositol 1,4,5-trisphosphate receptor 2 (Ip3r2-/-) to demonstrate that Ca2+ signalling is crucial for the post-transcriptional regulation of IL-25 production in tuft cells. The authors further demonstrated that the absence of IL-17RB expression in tuft cells led to chronic IL-25 exposure and hypo responsiveness in ILC2s, characterized by reduced proliferative capacity and altered expression of activation markers. This state of hypo responsiveness was also observed under conditions of prolonged exposure to elevated levels of IL-25, such as chronic succinate treatment and helminth infections. However, using an ILC2 intrinsic DREADD system (designer receptor exclusively activated by designer drug) (Il5-cre; TgCAG-lsl-Gq-DREADD), the authors showed that synergistic stimulation could overcome this hyporesponsive state. Collectively, this preprint addresses several central questions in the field of mucosal immunology and extends our understanding of tuft cell-ILC2 interactions both at steady state and during chronic helminth infections.

Limitations

• The authors used ILC2 activation as a surrogate read out for IL-25 production, direct measurement IL-25 protein level from tissues would strengthen the results.

• The authors nicely demonstrate succinate can indue calcium flux in tuft cells. It would be interesting to see if co-stimulation with IL-25 can alter calcium flux in tuft cells treated with succinate. This could add more evidence that signalling through IL-17RB limits tuft cell activation.

• It would be great to pair absolute cell counts with the percentages of cytokine positive and Ki67 positive cells in all experiments. It would illuminate whether the hypo proliferative state of the ILC2 is due to reaching a critical mass within the tissue or an ILC2-intrinsic effect.

• Within the literature, ILC2 have been demonstrated to undergo inter-organ trafficking following strong stimulus. Stimulus with either the DREADD system or systemic NMU treatments could therefore stimulate Ki67 expression and migration of ILC2 from distal tissues to the gut. Additional analysis of ILC2 activation in other tissues paired with a method to distinguish circulatory ILC2 from gut ILC2 would strengthen the results that secondary stimulus overcomes this hypo proliferative state in the intestine.

• Other groups have also described negative regulators for the ILC2-Tuft cell circuit (eg. PDG2 and A20). Analysis of these other regulators in the setting of tuft cell IL17RB depletion would reveal if any or these regulators have overlapping functions.

Significance/Novelty

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

The authors demonstrate that although tuft cells constitutively express Il25 mRNA, the production of IL-25 protein is regulated by IP3R2-dependent calcium flux. They further show that tuft cell expression of the receptor IL-17RB acts as a novel mechanism to limit IL-25 availability, serving as a novel brake on the ILC2-tuft cell circuit. This regulatory mechanism ensures controlled interaction between tuft cells and ILC2s, as ILC2s will enter a hypo-proliferative state after prolonged exposure to IL-25.

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

This work extends our understanding of the influence of intestinal epithelial cells on resident immune cells, identifying tuft cells as key regulators of ILC2 through an IL-25 dependent negative feedback loop that prevents overactivation of the immune response. It also demonstrates that while ILC2s can enter a hypo proliferative state as a natural adaptation to chronic stimulation, they may not enter full exhaustion, as synergistic signalling pathways can reverse this state. Therefore, researchers should account for microbiome-induced basal state activity to determine whether a particular stimulus can elicit biological responses.

Credit

Reviewed by Kyle Burrows (University of Toronto) 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 interests in relation to their involvement in the review.