Low-grade intestinal inflammation two decades after pelvic radiotherapy.

BACKGROUND: Radiotherapy is effective in the treatment of cancer but also causes damage to non-cancerous tissue. Pelvic radiotherapy may produce chronic and debilitating bowel symptoms, yet the underlying pathophysiology is still undefined. Most notably, although pelvic radiotherapy causes an acute intestinal inflammation there is no consensus on whether the late-phase pathophysiology contains an inflammatory component or not. To address this knowledge gap, we examined the potential presence of a chronic inflammation in mucosal biopsies from irradiated pelvic cancer survivors. METHODS: We biopsied 24 cancer survivors two to 20 years after pelvic radiotherapy, and four non-irradiated controls. Using tandem mass tag (TMT) mass spectrometry and mRNA sequencing (mRNA-seq), we charted proteomic and transcriptomic profiles of the mucosal tissue previously exposed to a high or a low/no dose of radiation. Changes in the immune cell populations were determined with flow cytometry. The integrity of the protective mucus layers were determined by permeability analysis and 16S rRNA bacterial detection. FINDINGS: 942 proteins were differentially expressed in mucosa previously exposed to a high radiation dose compared to a low radiation dose. The data suggested a chronic low-grade inflammation with neutrophil activity, which was confirmed by mRNA-seq and flow cytometry and further supported by findings of a weakened mucus barrier with bacterial infiltration. INTERPRETATION: Our results challenge the idea that pelvic radiotherapy causes an acute intestinal inflammation that either heals or turns fibrotic without progression to chronic inflammation. This provides a rationale for exploring novel strategies to mitigate chronic bowel symptoms in pelvic cancer survivors. FUNDING: This study was supported by the King Gustav V Jubilee Clinic Cancer Foundation (CB), The Adlerbertska Research Foundation (CB), The Swedish Cancer Society (GS), The Swedish State under the ALF agreement (GS and CB), Mary von Sydow's foundation (MA and VP).

A Preparatory Study for a Randomized Controlled Trial of Dietary Fiber Intake During Adult Pelvic Radiotherapy.

Background: Patients undergoing pelvic radiotherapy are often advised to omit fiber-rich foods from their diet to reduce the adverse effects of treatment. Scientific evidence supporting this recommendation is lacking, and recent studies on animals and humans have suggested that there is a beneficial effect of dietary fiber for the alleviation of symptoms. Randomized controlled studies on dietary fiber intake during pelvic radiotherapy of sufficient size and duration are needed. As preparation for such a large-scale study, we evaluated the feasibility, compliance, participation rate, and logistics and report our findings here in this preparatory study. Methods: In this preparatory study of a fiber intervention trial, Swedish gynecological cancer patients scheduled for radiotherapy were recruited between January 2019 and August 2020. During the intervention, the participants filled out questionnaires and used an application. They also consumed a fiber supplement at first in powder form, later in capsules. Blood- and fecal samples were collected. The study is registered in clinicaltrials.gov (https://clinicaltrials.gov/ct2/show/NCT04534075?cond=fidura&draw=2&rank=1). Results: Among 136 approached patients, 57 started the study and the participation rate for primary outcomes was 63% (third blood sample) and 65% (third questionnaire). Barely half of the participants provided fecal samples. Providing concise and relevant information to the patients at the right time was crucial in getting them to participate and stay in the study. The most common reasons for declining participation or dropping out were the expected burden of radiotherapy or acute side effects. Tailoring the ambition level to each patient concerning the collection of data beyond the primary endpoints was an important strategy to keep the dropout rate at an acceptable level. Using capsules rather than psyllium in powder form made it much easier to document intake and to create a control group. During the course of the preparatory study, we improved the logistics and for the last 12 participants included, the participation rate was 100% for the earliest primary outcome. Conclusion: A variety of adjustments in this preparatory study resulted in an improved participation rate, which allowed us to set a final protocol and proceed with the main study.

Dietary Fiber and the Hippocampal Neurogenic Niche in a Model of Pelvic Radiotherapy.

We sought to determine whether radiation to the colorectum had an impact on parameters of hippocampal neurogenesis and, if so, whether it could be modulated by a fiber-rich diet. Male C57BL/6J mice were fed a diet containing bioprocessed oat bran or a fiber-free diet, starting two weeks before colorectal irradiation with 4 fractions of 8 Gray or sham-irradiation. Diets were then continued for 1, 6 or 18 weeks, whereafter parameters of hippocampal neurogenesis were analyzed and correlated to serum cytokine levels. No statistically significant changes in neuronal markers or cell proliferation were found at one week post-irradiation. Six weeks post-irradiation there was a decreased cell proliferation in the subgranular zone that appeared slightly more pronounced in irradiated animals on a fiber-free diet and increased numbers of immature neurons per mm2 dentate gyrus in the irradiated mice, with a statistically significant increase in mice on a fiber-rich diet. Microglial abundancy was similar between all groups. 18 weeks post-irradiation, a fiber-free diet had reduced the number of immature neurons, whereas irradiation resulted in an increase. Despite this, the population of mature neurons was stable. Analysis of serum cytokines revealed a negative correlation between MIP1-α and the number of immature neurons one week after irradiation, regardless of diet. Our findings show that pelvic radiotherapy has the potential to cause a long-lasting impact on hippocampal neurogenesis, and dietary interventions may modulate this impact. More in-depth studies on the relationship between irradiation-induced intestinal injury and brain health are warranted.

Role of dietary fiber in safeguarding intestinal health after pelvic radiotherapy.

PURPOSE OF REVIEW: Damage to healthy bowel tissue during pelvic radiotherapy can produce devastating and life-long changes in bowel function. The surging interest in microbiota and its importance for our wellbeing has generated a bulk of research highlighting how the food we consume impacts bowel health and disease. Dietary fiber is known to promote bowel health, yet there is a limited number of studies on dietary fiber in connection to pelvic radiotherapy. Here, we review some of the literature on the subject and present the most recent publications in the field. RECENT FINDINGS: Advice given concerning dietary fiber intake during and after pelvic radiotherapy are inconsistent, with some clinics suggesting a decrease in intake and others an increase. Recent animal studies provide a solid support for a protective role of dietary fiber with regards to intestinal health after pelvic radiotherapy, mainly through its impact on the microbiota. No clinical study has yet provided unambiguous evidence for a similar function of dietary fiber in humans undergoing pelvic radiotherapy. SUMMARY: There is a lack of evidence behind the dietary advice given to cancer survivors suffering from radiation-induced bowel dysfunction, and high-quality and well powered studies with long follow-up times are needed.

A Fiber-Rich Diet and Radiation-Induced Injury in the Murine Intestinal Mucosa.

Dietary fiber is considered a strong intestinal protector, but we do not know whether dietary fiber protects against the long-lasting mucosal damage caused by ionizing radiation. To evaluate whether a fiber-rich diet can ameliorate the long-lasting pathophysiological hallmarks of the irradiated mucosa, C57BL/6J mice on a fiber-rich bioprocessed oat bran diet or a fiber-free diet received 32 Gray in four fractions to the distal colorectum using a linear accelerator and continued on the diets for one, six or 18 weeks. We quantified degenerating crypts, crypt fission, cell proliferation, crypt survival, macrophage density and bacterial infiltration. Crypt loss through crypt degeneration only occurred in the irradiated mice. Initially, it was most frequent in the fiber-deprived group but declined to levels similar to the fiber-consuming group by 18 weeks. The fiber-consuming group had a fast response to irradiation, with crypt fission for growth or healing peaking already at one week post-irradiation, while crypt fission in the fiber-deprived group peaked at six weeks. A fiber-rich diet allowed for a more intense crypt cell proliferation, but the recovery of crypts was eventually lost by 18 weeks. Bacterial infiltration was a late phenomenon, evident in the fiber-deprived animals and intensified manyfold after irradiation. Bacterial infiltration also coincided with a specific pro-inflammatory serum cytokine profile. In contrast, mice on a fiber-rich diet were completely protected from irradiation-induced bacterial infiltration and exhibited a similar serum cytokine profile as sham-irradiated mice on a fiber-rich diet. Our findings provide ample evidence that dietary fiber consumption modifies the onset, timing and intensity of radiation-induced pathophysiological processes in the intestinal mucosa. However, we need more knowledge, not least from clinical studies, before this finding can be introduced to a new and refined clinical practice.

Dietary Oat Bran Reduces Systemic Inflammation in Mice Subjected to Pelvic Irradiation.

Patients undergoing radiotherapy to treat pelvic-organ cancer are commonly advised to follow a restricted fiber diet. However, reducing dietary fiber may promote gastrointestinal inflammation, eventually leading to deteriorated intestinal health. The goal of this study was to evaluate the influence of dietary fiber on radiation-induced inflammation. C57BL/6J male mice were fed a High-oat bran diet (15% fiber) or a No-fiber diet (0% fiber) and were either irradiated (32 Gy delivered in four fractions) to the colorectal region or only sedated (controls). The dietary intervention started at 2 weeks before irradiation and lasted for 1, 6, and 18 weeks after irradiation, at which time points mice were sacrificed and their serum samples were assayed for 23 cytokines and chemokines. Our analyses show that irradiation increased the serum cytokine levels at all the time points analyzed. The No-fiber irradiated mice had significantly higher levels of pro-inflammatory cytokines than the High-oat irradiated mice at all time points. The results indicate that a fiber-rich oat bran diet reduces the intensity of radiation-induced inflammation, both at an early and late stage. Based on the results, it seems that the advice to follow a low-fiber diet during radiotherapy may increase the risk of decreased intestinal health in cancer survivors.

Long-term mucosal injury and repair in a murine model of pelvic radiotherapy.

Chronic intestinal injury after pelvic radiotherapy affects countless cancer survivors worldwide. A comprehensive understanding of the long-term injury dynamics is prevented in available animal models. With linear accelerators that are used to treat cancer in patients, we irradiated a small volume encompassing the colorectum in mice with four fractions of 8 Gy per fraction. We then determined the long-term dynamics of mucosal injury, repair, and the duration of inflammation. We show that crypt fission, not cell proliferation, is the main long-term mechanism for rescuing crypt density after irradiation, and provides a potentially wide window for clinical interventions. Persisting macrophage aggregations indicate a chronic mucosal inflammation. A better understanding as to how crypt fission is triggered and why it fails to repair fully the mucosa may help restore bowel health after pelvic radiotherapy. Moreover, anti-inflammatory interventions, even if implemented long after completed radiotherapy, could promote bowel health in pelvic cancer survivors.

Targeting Filamin A Reduces Macrophage Activity and Atherosclerosis.

BACKGROUND: The actin-binding protein FLNA (filamin A) regulates signal transduction important for cell locomotion, but the role of macrophage-specific FLNA during atherogenesis has not been explored. METHODS: We analyzed FLNA expression in human carotid atherosclerotic plaques by immunofluorescence. We also produced mice with Flna-deficient macrophages by breeding conditional Flna-knockout mice ( Flna o/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter ( LC). Atherosclerosis in vivo was studied by transplanting bone marrow from male Flna o/fl/ LC mice to atherogenic low-density lipoprotein receptor-deficient ( Ldlr-/-) mice; and by infecting Flna o/fl and Flna o/fl/ LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Furthermore, C57BL/6 mice were infected with AdPCSK9 and then treated with the calpain inhibitor calpeptin to inhibit FLNA cleavage. RESULTS: We found that macrophage FLNA expression was higher in advanced than in intermediate human atherosclerotic plaques. Flna o/fl/ LC macrophages proliferated and migrated less than controls; expressed lower levels of phosphorylated AKT and ERK1/2; exhibited reduced foam cell formation and lipid uptake; and excreted more lipids. The deficiency of Flna in macrophages markedly reduced the size of aortic atherosclerotic plaques in both Ldlr-/-BMT: Flnao/fl/LC and AdPCSK9-infected Flna o/fl/ LC mice. Intima/media ratios and numbers of CD68-positive macrophages in atherosclerotic plaques were lower in Flna-deficient mice than in control mice. Moreover, we found that STAT3 interacts with a calpain-cleaved carboxyl-terminal fragment of FLNA. Inhibiting calpain-mediated FLNA cleavage with calpeptin in macrophages reduced nuclear levels of phosphorylated STAT3, interleukin 6 secretion, foam cell formation, and lipid uptake. Finally, calpeptin treatment reduced the size of atherosclerotic plaques in C57BL/6 mice infected with AdPCSK9. CONCLUSIONS: Genetic inactivation of Flna and chemical inhibition of calpain-dependent cleavage of FLNA impaired macrophage signaling and function, and reduced atherosclerosis in mice, suggesting that drugs targeting FLNA may be useful in the treatment of atherosclerosis.

Blocking the Cleavage of Filamin A by Calpain Inhibitor Decreases Tumor Cell Growth.

BACKGROUND/AIM: Filamin A (FLNA) is the most abundant and widely expressed isoform of filamin in human tissues. It is cleaved by calpain at the hinge 1 and 2 domains, producing a 90-kDa carboxyl-terminal fragment (FLNACT). Recently, it has been shown that FLNACT mediates cell signaling and transports transcription factors into the cell nucleus. However, the significance of cleavage of FLNA by calpain has not been studied in cancer cell growth. Calpeptin is a chemical inhibitor of both calpain 1 and 2 that cleaves FLNA. In this study, we questioned if inhibiting calpain using calpeptin would decrease tumor cell proliferation, migration, invasion, and colony formation. MATERIALS AND METHODS: Human melanoma (A7), prostate cancer (PC3), mouse fibrosarcoma (T241) and endothelial (MS1) cells were assayed for proliferation, migration, invasion and colony formation after treatment with calpeptin. Cell lysates were immunoblotted for FLNA and FLNACT Results: Calpeptin treatment of these cells resulted in a decreased production of FLNACT Calpeptin-treated human and mouse tumor cells displayed impaired proliferation, migration, and colony formation. CONCLUSION: These data suggest that the cleavage of FLNA by calpain is an important cellular event in the regulation of tumor cell growth.