"Involvement of enteroendocrine cells in the intestinal immune response following ingestion of pathogenic bacteria"

President of the jury :                       

• Christine Coustau

Rapporteurs :                     

• Pauline Speder
• Cédric Pollesselo                         

Examiners

• Renata Matos

Thesis Director :

• Armel Gallet
• Dani Osman   

Abstract :

The gut has emerged as an attracting model for studying the mechanisms involved in tissue regeneration and innate immunity. During my thesis, I used Drosophila melanogaster, a suitable genetic model, to investigate the intestinal immune response and the production of enteroendocrine hormones.

Given the substantial quantity of food ingested over a lifespan, the likelihood of ingesting food contaminated by pathogenic bacteria is high. In the first part of my thesis, I focused on the intestinal innate immune response within the Drosophila larva following the ingestion of pathogenic bacteria. We uncovered a pylorus-like structure situated in the anterior midgut and demonstrated that its opening and closing are regulated by the enteroendocrine peptide DH31. We also characterized its role in the detection and elimination of pathogenic bacteria. Remarkably, we observed that as early as 15 minutes after ingesting pathogenic bacteria (both Gram-positive and Gram-negative), the pylorus closed, effectively obstructing the pathogenic bacteria within the anterior section of the intestine. We elucidated that this closure resulted from enterocytes producing reactive oxygen species (ROS), which accumulate in the intestinal lumen, in response to pathogenic bacteria. These ROS subsequently bound to the TRPA1 ion channel receptor located in DH31-expressing enteroendocrine cells surrounding the pylorus. We then demonstrated that this interaction triggered the release of the DH31 neuropeptide, prompting the closure of the pylorus and, consequently, the containment of pathogenic bacteria within the anterior part of the intestine. Lastly, we demonstrated that this blockade was essential to activate the innate immune pathway IMD, enabling the production of antimicrobial peptides. These peptides, in turn, effectively eliminate the bacteria trapped in the anterior midgut, ensuring the survival of the organism. Interestingly, the mammalian counterpart of DH31, CGRP (Calcitonin Gene-Related Peptide), has been identified in enteroendocrine cells located in the pylori of rat and pigs. Intriguingly, I have illustrated that the ingestion of the human orthologs of DH31, hCGRP, had the capability to induce the closure of the pylorus in Drosophila larva. Hence, our work shed lights on a pylorus-like structure within the Drosophila larval gut, which could serve as a model for investigating the function and roles of mammalian pylori. In the second part of my thesis, I investigated alterations in the population of enteroendocrine cells following ingestion of Bacillus thuringiensis (Bt)-based bioinsecticides. These bioinsecticides are offered as an eco-friendly alternative to chemical pesticides and are widely used to combat crop pests. Previous research conducted by my thesis laboratory had demonstrated that Bt Cry1A toxins, designed to eliminate Lepidopteran larvae, led to an excess of enteroendocrine cells in the intestine of adult Drosophila melanogaster, a dipteran species normally not targeted by Bt Cry1A toxins. Our findings revealed that a single exposure to Bt at an environmentally relevant dosage was sufficient to increase the number of enteroendocrine cells in the posterior segment of the Drosophila midgut, with this effect persisting up to day 7 post-infection, followed by a return to normal levels by day 10. Furthermore, we observed a continuous increase in enteroendocrine cells up to day 14, which was succeeded by a plateau phase extending until day 31 during chronic infection. Subsequently, I identified that the surplus enteroendocrines cells specifically expressed the neuropeptide Allatostatin A, which plays a role in regulating food intake and sleep.

Keywords :

Drosophila melanogaster, enteroendocrine cells, opportunistic bacteria, innate immune response, ROS, DH31/CGRP