Biological Effects of Abdominal Irradiation on Intestinal Barrier Breakdown Identified By Second-Generation Probiotic, LR-IL-22
Authors: Espinal Alexis Marie , Epperly Michael , Mukherjee Amitava , Shields Donna , Hou Wen , Fisher Renee , van Pijkeren Jan-Peter , Patel Ravi , Islam Naveed , Greenberger Joel S 
Published: February 2021

Toxic effects of total body irradiation and whole abdomen irradiation include breakdown of the intestinal barrier function and bacterial translocation into the abdominal lymphatic and systemic vascular circulations. Therapeutic effects of countermeasures against intestinal radiation damage have relied upon serum assays for bacterial endotoxin and citrulline, biomarkers of relatively late effects in barrier dysfunction. Intestinal barrier breakdown effects on mesenteric lymph nodes and spleen have been incompletely documented.
In the present experiments, we gavaged C57BL/6NTac female mice one hour before irradiation (19.75 Gy whole abdomen) with 100 microliters containing 109 second-generation probiotic, Lactobacillus reuteri-Interleukin-22 (LR-IL-22) containing transgenes for both mammalian IL-22 and Rapamycin resistance (Alexander LM, et. al., Applied and Environmental Microbiology 85(10): e02335-18, 2019), LR alone, or sham-gavage. Twenty-four hours later, Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood were collected at serial times after gavage and assayed for growth of Rapamycin-resistant bacteria, and for relative compositions of immunocytes in mesenteric lymph nodes spleen and blood including: CD3, CD19, CD8, CD45, NK1.1, CD25, Foxp3, CD103, and PD-1+ lymphocytes.
Compared to non-irradiated, non-gavaged mice, animals receiving LR or LR-IL-22 showed no significant changes in weight or length of the spleen or lymph nodes 24 hr after gavage. There was an increase in PD-1 expression in the spleen of the mice gavaged with LR-IL-22. There were no other significant changes in the composition of immunocytes in abdominal lymph nodes, or spleen and detectable second-generation probiotic growth in these organs. At 24 hr after irradiation, abdominal irradiated mice demonstrated a significant loss of weight and length of the spleen (p &lt; 0.05) as well as decreased lymphocytes in the blood (p &lt; 0.05). Mice gavaged with LR-IL-22 and irradiated to 19.75 Gy had a significant increase in lymphocytes and red blood cells compared to irradiation only (p &lt; 0.05). There was no significant change in the size or weight of the lymph nodes. In the lymph nodes there was an increase in CD8+ cells after irradiation while the LR-IL-22 gavaged mice had an increase in PD-1 expression. There was no detectable growth of Rapamycin resistant bacteria in non-gavaged animals. Mice gavaged with LR-IL-22, but not LR showed decreased biomarkers of intestinal barrier breakdown. Thus, gavage of genetically labeled second-generation probiotics represents a sensitive assay system for irradiation changes in the intestinal barrier function, translocation of bacteria into the abdominal lymphatic system, and therapeutic effect of enteric delivered radiation countermeasures.
Supported by U19-AI1068021 Grant.

Disclosures
No relevant conflicts of interest to declare.