Abstract Cellular senescence is involved in the development of pulmonary fibrosis as well as in lung tissue repair and regeneration. Therefore, a strategy of removal of senescent cells by senolytic drugs may not produce the desired therapeutic result. Previously we reported that tyrosine kinase Fgr is upregulated in ionizing irradiation-induced senescent cells. Inhibition of Fgr reduces the production of profibrotic proteins by radiation-induced senescent cells in vitro; however, a mechanistic relationship between senescent cells and radiation-induced pulmonary fibrosis (RIPF) has not been established. We now report that senescent cells from the lungs of mice with RIPF, release profibrotic proteins for target cells and secrete chemotactic proteins for marrow cells. The Fgr inhibitor TL02-59, reduces this release of profibrotic chemokines from the lungs of RIPF mice, without reducing numbers of senescent cells. In vitro studies demonstrated that TL02-59 abrogates the upregulation of profibrotic genes in target cells in transwell cultures. Also, protein arrays using lung fibroblasts demonstrated that TL02-59 inhibits the production of chemokines involved in the migration of macrophages to the lung. In thoracic-irradiated mice, TL02-59 prevents RIPF, significantly reduces levels of expression of fibrotic gene products, and significantly reduces the recruitment of CD11b+ macrophages to the lungs. Bronchoalveolar lavage (BAL) cells from RIPF mice show increased Fgr and other senescent cell markers including p16. In human idiopathic pulmonary fibrosis (IPF) and in RIPF, Fgr, and other senescent cell biomarkers are increased. In both mouse and human RIPF, there is an accumulation of Fgr-positive proinflammatory CD11b+ macrophages in the lungs. Thus, elevated levels of Fgr in lung senescent cells upregulate profibrotic gene products, and chemokines that might be responsible for macrophage infiltration into lungs. The detection of Fgr in senescent cells that are obtained from BAL during the development of RIPF may help predict the onset and facilitate the delivery of medical countermeasures.
Background/Aim: There is concern that people who had COVID-19 will develop pulmonary fibrosis. Using mouse models, we compared pulmonary inflammation following injection of the spike protein of SARS-CoV-2 (COVID-19) to radiation-induced inflammation to demonstrate similarities between the two models. SARS-CoV-2 (COVID-19) induces inflammatory cytokines and stress responses, which are also common to ionizing irradiation-induced acute pulmonary damage. Cellular senescence, which is a late effect following exposure to SARS-CoV-2 as well as radiation, was investigated. Materials and Methods: We evaluated the effect of SARS-CoV-2 spike protein compared to ionizing irradiation in K18-hACE2 mouse lung, human lung cell lines, and in freshly explanted human lung. We measured reactive oxygen species, DNA double-strand breaks, stimulation of transforming growth factor-beta pathways, and cellular senescence following exposure to SARS-CoV-2 spike protein, irradiation or SARS-COV-2 and irradiation. We also measured the effects of the antioxidant radiation mitigator MMS350 following irradiation or exposure to SARS-CoV-2. Results: SARS-CoV-2 spike protein induced reactive oxygen species, DNA double-strand breaks, transforming growth factor-β signaling pathways, and senescence, which were exacerbated by prior or subsequent ionizing irradiation. The water-soluble radiation countermeasure, MMS350, reduced spike protein-induced changes. Conclusion: In both the SARS-Co-2 and the irradiation mouse models, similar responses were seen indicating that irradiation or exposure to SARS-CoV-2 virus may lead to similar lung diseases such as pulmonary fibrosis. Combination of irradiation and SARS-CoV-2 may result in a more severe case of pulmonary fibrosis. Cellular senescence may explain some of the late effects of exposure to SARS-CoV-2 spike protein and to ionizing irradiation.
Abstract Acute Radiation Syndrome and the multiorgan failure from delayed effects of acute radiation exposure present challenging consequences of radiation terrorism or a radiological accident. Recent research suggests that radiation-induced cellular senescence plays an important role in radiation-induced pulmonary fibrosis (RIPF), and that clearance of senescent cells (SCs) could be an effective therapeutic strategy. However, the identification and targeted removal of only senescent cells using drugs have been difficult. Here we have established a bone-marrow stromal cell line from a tdTOMp16+ mouse. We show that 5 Gy irradiation induces ~9% cellular senescence in tdTOMp16+ stromal line after 10 days and can be isolated as a pure population of red tdTOMp16+ cells by FACS sorting. To confirm that these irradiated and then sorted red cells are senescent, we cultured them as single cells in 96-well plates and followed for four weeks. None of the 960 irradiated red cells we plated divided after 2 weeks, 137 of these cells remained intact and assumed large and flat cellular morphology. In contrast, 50 of 800 cells that were irradiated but didn’t turn red, divided, as did 104 of 560 nonirradiated control cells (0 Gy) divided. There was significant upregulation of senescent cell markers including SA-ß-gal, p16, and p21 in the irradiated and sorted red cells when compared to irradiated nonred cells. Sorted irradiated red, non-irradiated, or irradiated non-red cells were placed on the top well of transwells. There was a significant induction of fibrotic genes Ctgf, Tgf- ß, collagen 1a, and collagen 3 in C57/B6 stromal target cells in the bottom layer by irradiated red cells, but not the other cell populations irradiated non-red. Thus, radiation-induced biomarkers of fibrosis are induced by senescent cells. Citation Format: Amitava Mukherjee, Michael Epperly, Donna Shields, Wen Hou, Renee Fisher, Diala Hamade, Joel S. Greenberger. Radiation-induced and FACS-sorted senescent tdTOMp16+ cells upregulate profibrotic genes in C57BL/6 stromal target cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-025.
Abstract Ovarian cancer is the most lethal gynecological cancer worldwide with an estimated 152,000 deaths per year. Despite optimal management with radical cytoreductive surgery and subsequent platinum/taxane-based chemotherapy, most patients, will suffer recurrence within 18 months. Our laboratory has recently discovered a new therapeutic agent for intestinal radiation protection, namely the novel second-generation probiotic Lactobacillus reuteri (LR) genetically engineered to produce the radioprotective cytokine Interleukin-22 (IL-22) (Zhang et al. In Vivo, 34(1):39-50, 2020 Jan-Feb). To demonstrate that LR-IL-22 could protect the intestines from irradiation, we used three mouse models (total body irradiation (TBI), whole abdomen irradiation (WAI) and partial body irradiation (PBI)). For TBI, C57BL/6 mice were irradiated to 9.25 Gy to the entire body. For WAI, C57BL/6 mice were irradiated using a linear accelerator so that only the abdomen was irradiated to 19.75 Gy with the remainder of the body shielded from the irradiation. PBI was performed with the right rear leg shielded with the rest of the body irradiated to 15 Gy. In all three models the mice were gavaged 24 hours after irradiation with 1 × 109 LR-IL-22 cells. The mice were followed for development of either the hematopoietic syndrome (TBI) or gastrointestinal syndrome (WAI or PBI). In separate experiments we determined if mice irradiated as above had decreased irradiation induced inflammation using a Luminex assay on the intestine and blood plasma from mice sacrificed on days 0, 1, 2, 3, 5 and 7 following irradiation. We also determined whether intraoral gavage of LR-IL-22 24 hours prior to irradiation might protect the tumor in a mouse ovarian tumor model. Murine ovarian tumor cells 2F8-cis were injected intraperitoneally into Muc1 transgenic mice. Seventy-two hours later the mice were irradiated to 16 Gy WAI and followed for tumor growth. In the TBI model, mice treated with LR-IL-22 24 hours prior to irradiation had an increased survival of 80% compared to 0% in control irradiated mice (p = 0.0001). In the WAI model, mice treated with LR-IL-22 had a 40% survival following 19.75 Gy compared to 0% in the control irradiated group (p = 0. 0100). Following the PBI dose of 15 Gy, mice treated with LR-IL-22 had a 70% survival compared to 0% for the control irradiation only mice (p = 0.0006). Decreased expression of several inflammatory proteins such as TNF-α, IL-6 and IFN-γ (p = 0.0423, 0.0473 and 0.0024, respectively) was also detected in mice treated with LR-IL-22 24 hours prior to WAI compared to control irradiated mice. Furthermore, two weeks after injecting Muc1 transgenic mice with tumors, the nonirradiated mice had more than 200 small tumor nodules disseminated throughout the peritoneum while control WAI mice or mice treated with intraoral LR-IL-22 prior to WAI had no more than 10 tumor nodules. Hence, intraoral LR-IL-22 prior to chemoradiation may protect the intestines and result in increased survival of ovarian cancer patients. Citation Format: Diala Fatima Hamade, Renee Fisher, Wen Hou, Donna Shields, Michael W. Epperly, Joel S. Greenberger. LR-IL-22 protects the intestine to facilitate whole abdomen irradiation in ovarian cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-081.
