Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5

Abstract

Introduction: The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5).

Methods: Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels.

Results: Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation.

Conclusion: These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Keywords: GI-ARS; barrier function; defensins; intestine; irradiation; microbiome; tight junction.

Figure 1

Ionizing radiation down-regulates Paneth cell α-defensins, alters gut microbiota, increases gut permeability, and leads to endotoxemia. Adult mice were exposed to ionizing radiation (IR) or sham-treated (0 hour). Intestinal α-defensin expression (A-F), microbiota composition (G-I), mucosal permeability (J), and plasma LPS (K) were analyzed at varying times after irradiation (post-IR). (A-C) RNA preparations from the ileum and colon were analyzed for Defa5 (A, B) and Defa6 (C) mRNA by RT-qPCR. Zero-hour values represent the sham-treated group. Values are mean ± sem (n = 6); *p<0.05 for significant difference from “0 h” value. (D-F) Colonic luminal flushing from Sham-treated and irradiated mice at 24 hours after irradiation were analyzed for overall α-defensins (D), DEFA5 (E), and DEFA21 (F) by mass spectrometry. Values are mean ± sem (n = 6); **p<0.01, ***p<0.001 for significant difference from corresponding “Sham” values. (G-I) At varying times after irradiation, DNA preparations from colonic luminal flushing were analyzed for selected microbiota taxa by RT-qPCR. Results of Firmicutes/Bacteroidetes or F/B ratio (G), Enterobacteriaceae (H), and E. coli (I) by mass spectrometry. Zero-hour values represent the sham-treated group. Values are mean ± sem (n = 4); *p<0.05, **p<0.01 for significant difference from corresponding “0 h” values. (J) At varying times after irradiation, colonic mucosal permeability in vivo was evaluated by measuring the vascular-to-luminal flux of FITC-inulin. Zero-hour values represent the sham-treated group. Values are mean ± sem (n = 6); *p<0.05 for significant difference from corresponding “0 h” value. (K) Plasma LPS levels were measured at varying times after irradiation. Zero-hour values represent the sham-treated group. Values are mean ± sem (n = 6); *p<0.05 for significant difference from corresponding “0 h” value.

Figure 2

Prophylactic HD5 treatment attenuates radiation-induced intestinal dysbiosis. Adult mice were fed a liquid diet with vehicle (Veh-Sham & Veh-IR) or HD5 (HD5-Sham & HD5-IR) for 24 hours before sham-treatment (Sham) or irradiated (IR). At 24 hours after irradiation, the microbiota composition in colonic flushing was analyzed by 16S rRNA-sequencing and metagenomics. (A) The relative abundance of different phyla of bacteria. Data are derived from pooling all values within the group. The experiment was repeated once with similar results. (B) The Shannon Index was used to quantify α-diversity. (C) Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity analysis was performed to determine β-diversity. (D-G) Relative abundance of Enterobacteriaceae (D), E. coli (E), Lactobacillus (F), and Akkermansia (G) in different groups.

Figure 3

Prophylactic HD5 treatment attenuates radiation-induced colonic mucosal injury. Adult mice were fed a liquid diet with vehicle (Veh-Sham & Veh-IR) or HD5 (HD5-Sham & HD5-IR) for 24 hours before sham-treatment (Sham) or irradiated (IR). TJ and AJ integrity, mucosal permeability, and mucosal inflammatory responses were analyzed 24 hours after irradiation. (A, B) TJ integrity was assessed by immunofluorescence staining of colon cryosections for occludin and ZO-1 (green, occludin; red, ZO-1; blue, nucleus) and confocal microscopy. ZO-1 fluorescence density values are presented in panel (B). (C, D) AJ integrity was assessed by staining colon sections for E-cadherin and β-catenin (green, E-cadherin; red, β-catenin; blue, nucleus). β-catenin fluorescence density values are presented in panel (D). (E, F) Mucosal permeability in vivo was evaluated in the colon (E) and ileum (F) by measuring the vascular-to-luminal flux of FITC-inulin. Values are mean ± sem (n = 6); *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for significant difference between the indicated groups; “ns”, not significant. (G-L) At 24 hours after irradiation, total RNA prepared from the colon were analyzed for IL-1β (G), IL-6 (H), TNFα (I), Mcp1 (J), Cxcl1 (K), and Cxcl2 (L). Values are mean ± sem (n = 6); *p<0.05, **p<0.01, and ****p<0.0001 for significant difference between the indicated groups; “ns”, not significant.

Figure 4

HD5 administered at 24 hours after irradiation modulates altered gut microbiota composition. At 24 hours after sham treatment (Sham) or irradiation (IR), mice were fed a liquid diet with vehicle (Veh-Sham & Veh-IR) or HD5 (HD5-Sham & HD5-IR). After additional 24 hours, the microbiota composition in colonic flushing was analyzed by 16S rRNA-sequencing and metagenomics. (A) The relative abundance of different phyla of bacteria. Data are derived from pooling all values within the group. The experiment was repeated once with similar results. (B) The Shannon Index was used to quantify α-diversity. (C) Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity analysis was performed to determine β-diversity. (D-G) Relative abundance of Enterobacteriaceae (D), E. coli (E), Lactobacillus (F), and Akkermansia (G) in different groups.

Figure 5

HD5 feeding at 24 hours after irradiation mitigates colonic mucosal injury. At 24 hours after sham treatment (Sham) or irradiation (IR) mice were fed a liquid diet with vehicle (Veh-Sham & Veh-IR) or HD5 (HD5-Sham & HD5-IR). After additional 24 hours, TJ and AJ integrity, mucosal permeability, and mucosal inflammatory responses were analyzed. (A, B) TJ integrity was assessed by immunofluorescence staining of colon cryosections for occludin and ZO-1 (green, occludin; red, ZO-1; blue, nucleus) and confocal microscopy. ZO-1 fluorescence density values are presented in panel (B). (C, D) AJ integrity was assessed by staining colon sections for E-cadherin and β-catenin. β-catenin fluorescence density values are presented in panel (D). (E, F) Mucosal permeability in vivo was evaluated in the colon (E) and ileum (F) by measuring the vascular-to-luminal flux of FITC-inulin. Values are mean ± sem (n = 6); **p<0.01, ***p<0.001, and ****p<0.0001 for significant difference between the indicated groups; “ns” = not significant. (G-L) At 48 hours after irradiation (24 hours after start of HD5 treatment), total RNA preparations from the colon were analyzed for IL-1β (G), IL-6 (H), TNFα (I), Mcp1 (J), Cxcl1 (K), and Cxcl2 (L). Values are mean ± sem (n = 6); *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for significant difference between the indicated groups; “ns”, not significant.

Figure 6

Prevention and reversion of radiation-induced endotoxemia and systemic inflammation by HD5. (A-D) Adult mice were fed a liquid diet with vehicle (Veh-Sham & Veh-IR) or HD5 (HD5-Sham & HD5-IR) for 24 hours before sham treatment (Sham) or irradiated (IR). At 24 hours after irradiation, plasma LPS (A), TNFα (B), IL-6 (C), and IL-1β (D) were measured. Values are mean ± sem (n = 6); *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for significant difference between the indicated groups; “ns” = not significant. (E-H) At 24 hours after sham treatment (Sham) or irradiation (IR), mice were fed a liquid diet with vehicle (Sham-Veh & IR-Veh) or HD5 (Sham-HD5 & IR-HD5). After additional 24 hours, plasma LPS (E), TNFα (F), IL-6 (G), and IL-1β (H) were measured. Values are mean ± sem (n = 6); *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 for significant difference between the indicated groups; “ns”, not significant.

Figure 7

Graphic summary. 1) Radiation-induced Paneth cell dysfunction down-regulates α-defensin expression. 2) α-Defensin depletion leads to dysbiosis of gut microbiota, causing increased LPS production. 3) Radiation disrupts epithelial tight junctions leading to increased LPS translocation. 4) Increased LPS translocation results in endotoxemia. 5) Endotoxemia leads to systemic inflammation. 6) Human defensin-5 (HD-5) supplementation attenuates and mitigates radiation-induced microbiota dysbiosis, endotoxemia, and systemic inflammation.