Gut Microbiota‐Derived 3‐Hydroxybutyrate Blocks GPR43‐Mediated IL6 Signaling to Ameliorate Radiation Proctopathy

Abstract Radiation proctopathy (RP) is a common complication of radiotherapy for pelvic malignancies with high incidence. RP accompanies by microbial dysbiosis. However, how the gut microbiota affects the disease remains unclear. Here, metabolomics reveals that the fecal and serous concentrations of microbiota‐derived 3‐hydroxybutyrate (3HB) are significantly reduced in RP mice and radiotherapeutic patients. Moreover, the concentration of 3HB is negatively associated with the expression of proinflammatory IL6 that is increased along with the severity of radiation damage. 3HB treatment significantly downregulates IL6 expression and alleviates IL6‐mediated radiation damage. Irradiated cell‐fecal microbiota co‐culture experiments and in vivo assays show that such a radioprotection of 3HB is mediated by GPR43. Microbiome analysis reveals that radiation leads to a distinct bacterial community compared to untreated controls, in which Akkermansia muciniphila is significantly reduced in RP mice and radiotherapeutic patients and is associated with lower 3HB concentration. Gavage of A. muciniphila significantly increases 3HB concentration, downregulates GPR43 and IL6 expression, and ameliorates radiation damage. Collectively, these results demonstrate that the gut microbiota, including A. muciniphila, induce higher concentrations of 3HB to block GPR43‐mediated IL6 signaling, thereby conferring radioprotection. The findings reveal a novel implication of the gut‐immune axis in radiation pathophysiology, with potential therapeutic applications.


Supplementary figures and legends
(A) Endoscopy examination confirms that patients had RP lesions after radiotherapy.
Endoscopy examination showed subsequent changes in the rectal mucosa after a total irradiation dose of 50 Gy.The typical case revealed telangiectasis and active bleeding sites in the intestinal mucosa within the RP lesions (yellow arrow).
(B) Clinical score of mice before radiation and post-radiation at 6 and 8 weeks.Table S1.

Figure S1 .
Figure S1.The concentrations of 3HB in serum that significantly decreased in

Figure S2 .
Figure S2.Antibiotic treatment removes most of the bacteria from the intestine.

Figure S3 .
Figure S3.The concentration of many metabolites in feces is associated with

Figure S4 .
Figure S4.The production of gut microbiota-derived 3HB in feces plays a

Figure S5 .
Figure S5.Longitudinal follow-up of 3HB concentrations after antibiotic

Figure S6 .
Figure S6.The concentrations of 3HB of stool and serum that significantly
(C-D) Correlation between age and 3HB concentration in feces (C) and serum (D) sample derived from oncology patients after radiotherapy treatment.(E-F) The concentration of 3HB in feces (E) and serum (F) between oncology patients received with folfox and folfox/PD1 treatment.Data are representative of at least two biological replicates.Data are presented as the mean ± SEM.Animal samples were collected at 2, 4, and 6 weeks respectively after radiation treatment, N = 6 per group.Patient samples were collected one day after the completion of radiotherapy (RT), N = 20 for oncology patients.**P < 0.01 and no significance (ns) determined by the Student's t-test [(A), (B), (E), and (F)] and Spearman correlation [(C)and (D)].

(
H) Representative images of H&E and Masson immunostaining of the distal rectum.Insets are showed at a higher magnification on the right.(I) Histopathological changes evaluated by calculating RIS score.(J) Tumor numbers in colorectal tissue of each group.Data are representative of at least two biological replicates.Data are presented as the mean ± SEM.Samples were collected after 3HB treatment for 5 weeks.N = 6 per group.*P < 0.05, and **P < 0.01 determined by the Student's t-test [(B), (C), (D), (F), (G), (I), and (J)].

Figure S9 .
Figure S9.Reduced radiation-induced damage mediated by 3HB are impaired in

Figure S10 .
Figure S10.Western blot analysis of phosphorylated STAT3 in rectal tissue

Figure S13 .
Figure S13.The concentration of 3HB in feces is associated with specific gut

Figure
Figure S14. A. muciniphila plays a contributing role in accumulating 3HB levels

Figure S15 .
Figure S15.The viability of A. muciniphila in co-culture experiments.

Figure S16 .
Figure S16.Oral administration of A. muciniphila can colonize the intestine of

Figure S18 .
Figure S18.Examination of patients and mice after radiation therapy.
increase in collagen fibers 2 = Dense fibers were significantly increased, and the vessel wall was hyaline degeneration