Glia-mediated gut-brain cytokine signaling couples sleep to intestinal inflammation

Joint Public Review

Summary:

The authors sought to elucidate the mechanism by which infections increase sleep in Drosophila. Their work is important because it further supports the idea that the blood-brain barrier is involved in brain-body communication, and because it advances the field of sleep research. Using knock-down and knock-out of cytokines and cytokine receptors specifically in the endocrine cells of the gut (cytokines) as well as in the glia forming the blood-brain barrier (BBB) (cytokines receptors), the authors show that cytokines, upd2 and upd3, secreted by entero-endocrine cells in response to infections increase sleep through the Dome receptor in the BBB. They also show that gut-derived Allatostatin (Alst) A promotes wakefulness by inhibiting Alst A signaling that is mediated by Alst receptors expressed in BBB glia. Their results suggest there may be additional mechanisms that promote elevated sleep during gut inflammation.
The authors suggest that upd3 is more critical than upd2, which is not sufficiently addressed or explained. In addition, the study uses the gut's response to reactive oxygen molecules as a proxy for infection, which is not sufficiently justified. Finally, further verification of some fundamental tools used in this paper would further solidify these findings making them more convincing.

Strengths:

(1) The work addresses an important topic and proposes an intriguing mechanism that involves several interconnected tissues. The authors place their research in the appropriate context and reference related work, such as literature about sickness-induced sleep, ROS, the effect of nutritional deprivation on sleep, sleep deprivation and sleep rebound, upregulated receptor expression as a compensatory mechanism in response to low levels of a ligand, and information about Alst A.

(2) The work is, in general, supported by well-performed experiments that use a variety of different tools, including multiple RNAi lines, CRISPR, and mutants, to dissect both signal-sending and receiving sides of the signaling pathway.

(3) The authors provide compelling evidence that shows that endocrine cells from the gut are the source of the upd cytokines that increase daytime sleep, that the glial cells of the BBB are the targets of these upds, and that upd action causes the downregulation of Alst receptors in the BBB via the Jak/Stat pathways.

Weaknesses:

(1) There is a limited characterization of cell types in the midgut which are classically associated with upd cytokine production.

(2) Some of the main tools used in this manuscript to manipulate the gut while not influencing the brain (e.g., Voilà and Voilà + R57C10-GAL80), are not directly shown to not affect gene expression in the brain. This is critical for a manuscript delving into intra-organ communication, as even limited expression in the brain may lead to wrong conclusions.

(3) The model of gut inflammation used by the authors is based on the increase in reactive oxygen species (ROS) obtained by feeding flies food containing 1% H2O2. The use of this model is supported by the authors rather weakly in two papers (refs. 26 and 27 ): The paper by Jiang et al. (ref. 26) shows that the infection by Pseudomonas entomophila induces cytokine responses upd2 and 3, which are also induced by the Jnk pathway. In addition, no mention of ROS could be found in Buchon et al. (ref 27); this is a review that refers to results showing that ROS are produced by the NADPH oxidase DUOX as part of the immune response to pathogens in the gut. Thus, there is no strong support for the use of this model.

(4) Likewise, there is no support for the use of ROS in the food instead a direct infection by pathogenic bacteria. Furthermore, it is known that ROS damages the gut epithelium, which in turn induces the expression of the cytokines studied. Thus the effects observed may not reflect the response to infection. In addition, Majcin Dorcikova et al. (2023). Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila. Nat Commun. 2023 14(1):5908. doi: 10.1038/s41467-023-41540-y report that the feeding of adult flies with H2O2 results in neurodegeneration if associated with circadian clock defects. Thus, it would be important to discuss or present controls that show that the feeding of H2O2 does not cause neuronal damage.

(5) The novelty of the work is difficult to evaluate because of the numerous publications on sleep in Drosophila. Thus, it would be very helpful to read from the authors how this work is different and novel from other closely related works such as: Li et al. (2023) Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila. Cell Discov. 23;9(1):49. doi: 10.1038/s41421-023-00541-3.