The mechanism by which intestinal flora metabolites regulate ILC2s transformation and intestinal immunity through the metabolite-sensitive receptor Ffar2
Authors: Li Huimin , Han Liping , Ge Haiyan , Jia Menghan 
Published: September 2024

ABSTRACT

Objective:
The objective of this article was to investigate how the metabolites produced by the intestinal flora regulate the transformation of ILC2s and intestinal immunity via the receptor Ffar2, which is sensitive to metabolites.

Materials and Methods:
Forty male C57BL/6 mice with wild-type characteristics (6-7 weeks in age) and 20 FFAR2-/- mice were acquired from The Jackson Laboratory. The mice were kept in a controlled environment without any disease-causing agents, with the help of air conditioning and a 12-hour cycle of light and darkness. Throughout the experiment, every mouse was provided with unrestricted availability of both food and water. All protocols were performed following the Regulations of Animal Welfare and the recommendations for Animal Testing. According to the research protocol, the mice were categorized into three groups, each consisting of 20 mice. FFAR2-/- mice in the FFAR2-/- group were reared in conventional environments. Male C57BL/6 mice with wild-type characteristics were reared in conventional conditions in the wild-type group. In the group where wild-type mice were inhibited with Ffar2, a total of 20 mice with the wild-type phenotype were chosen to receive intraperitoneal injection of an FFAR2 antagonist at a dosage of 5 mg/kg per day for a continuous period of 3 weeks. qRT-PCR was used to detect the expression of FFAR2 mRNA in the mucosal tissue of the mouse colon. High-throughput sequencing was used to conduct an examination of metabolites in the intestinal tract of mice. Flow cytometry was used to test the quantity of ILC2s. ELISA was used to measure the levels of IL-5 and IL-13 in cellular mouse intestinal mucosal tissues. Flow cytometry was used to detect the quantity of CD4+ and CD8+ T cells. CCL20 and CCL25 expression was analyzed by Western blotting. The level of FFAR2 mRNA expression was higher in the wild-type group than the FFAR2-/- group (P < 0.05), while it was lower in the wild-type + Ffar2 inhibition group than the wild-type group (P < 0.05). No variations were observed in the composition of metabolites and levels of major SCFAs in the gut microbiota of mice across all groups (P > 0.05). The quantity of CRTH2+ and ST2+ cells in the wild-type category exceeded that in the FFAR2-/- category (P < 0.05), whereas the quantity of CRTH2+ and ST2+ cells in the wild-type + Ffar2 inhibition category was lower than that in the wild-type category (P < 0.05). The concentrations of IL-5 and IL-13 were higher in the wild-type group compared with the FFAR2-/- group (P < 0.05), while the wild-type + Ffar2 inhibition group exhibited lower levels of IL-5 and IL-13 than the wild-type group (P < 0.05). The count of CD4+ T cells and CD8+ T cells in the wild-type group showed an increase in comparison to the FFAR2-/- group (P < 0.05), whereas the count of CD4+ T cells and CD8+ T cells in the wild-type + Ffar2 inhibition group exhibited a decrease in comparison to the wild-type group (P < 0.05). The levels of protein expression for CCL20 and CCL25 were higher in the wild-type group compared with the FFAR2-/- group (P < 0.05), whereas the wild-type + Ffar2 inhibition group exhibited lower protein expression of CCL20 and CCL25 than the wild-type group (P < 0.05).

Conclusion:
Ffar2 has a significant regulatory function in the conversion of ILC2s, consequently impacting the immune response in the intestines. Ffar2, a crucial receptor for metabolites produced by the intestinal flora, plays a vital function in controlling the conversion of ILC2s and the overall immune response in the intestines.