No abstract

Neutrophilic inflammation is essential for defending against invading pathogens, but can also be detrimental in many clinical settings. The hematopoietic-specific small Rho-GTPase Rac2 regulates multiple pathways that are essential for neutrophil activation, including adhesion, migration, degranulation and production of reactive oxygen species. This study tested the hypothesis that partially suppressing rac2 in zebrafish neutrophils by using a microRNA (miRNA) would inhibit neutrophil migration and activation, which would reduce the immunological damage caused by systemic inflammation. We have generated a transgenic zebrafish line that overexpresses microRNA-722 (miR-722) in neutrophils. Neutrophil motility and chemotaxis to tissue injury or infection are significantly reduced in this line. miR-722 downregulates the transcript level of rac2 through binding to seed-matching sequence in the rac2 3′UTR. Furthermore, miR-722-overexpressing larvae display improved outcomes in both sterile and bacterial systemic models, which correlates with a robust upregulation of the anti-inflammatory cytokines in the whole larvae and isolated neutrophils. Finally, an miR-722 mimic protects zebrafish from lethal lipopolysaccharide challenge. Together, these results provide evidence for and the mechanism of an anti-inflammatory miRNA that restrains detrimental systemic inflammation.

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EECs). EECs can communicate nutritional information to the nervous system, but similar mechanisms for microbial information are unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda specifically activates EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increases intestinal motility in an EEC-dependent manner. Microbial, pharmacological, or optogenetic activation of Trpa1 + EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons through 5-HT. We identified a distinct subset of indole derivatives of tryptophan catabolites produced by E. tarda that potently activate zebrafish EEC Trpa1 signaling and also directly activate human and mouse Trpa1. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to specific microbial signals.

No abstract

Transcription factors play important roles in development of the intestinal epithelium and its ability to respond to endocrine, nutritional, and microbial signals. Hepatocyte Nuclear Factor 4 (Hnf4) family nuclear receptors are liganded transcription factors that are critical for the development and function of multiple digestive organs in vertebrates, including the intestinal epithelium. Zebrafish have three Hnf4 homologs, of which, hnf4a was previously shown to mediate intestinal responses to microbiota in zebrafish larvae. To discern the functions of other Hnf4 family members in zebrafish development and intestinal function, we created and characterized mutations in hnf4g and hnf4b. We addressed the possibility of genetic redundancy amongst these factors by creating double and triple mutants which showed different rates of survival, including apparent early lethality in hnf4a;hnf4b double mutants and triple mutants. RNA sequencing performed on digestive tracts from single and double mutant larvae revealed extensive changes in intestinal gene expression in hnf4a mutants that were amplified in hnf4a;hnf4g mutants, but limited in hnf4g mutants. Changes in hnf4a and hnf4a;hnf4g mutants were reminiscent of those seen in mice including decreased expression of genes involved in intestinal function and increased expression of cell proliferation genes, and were validated using transgenic reporters and EdU labeling in the intestinal epithelium. Gnotobiotics combined with RNA sequencing also showed hnf4g has subtler roles than hnf4a in host responses to microbiota. Overall, phenotypic changes in hnf4a single mutants were strongly enhanced in hnf4a;hnf4g double mutants, suggesting a conserved partial genetic redundancy between hnf4a and hnf4g in the vertebrate intestine.