Werner Olipitz

Unifying roles for regulatory T cells and inflammation in cancer

Activities of CD4+ regulatory (TREG) cells restore immune homeostasis during chronic inflammatory disorders. Roles for TREG cells in inflammation‐associated cancers, however, are paradoxical. It is widely believed that TREG function in cancer mainly to suppress protective anticancer responses. However, we demonstrate here that TREG cells also function to reduce cancer risk throughout the body by efficiently downregulating inflammation arising from the gastrointestinal (GI) tract. Building on a “hygiene hypothesis” model in which GI infections lead to changes in TREG that reduce immune‐mediated diseases, here we show that gut bacteria‐triggered TREG may function to inhibit cancer even in extraintestinal sites. Ability of bacteria‐stimulated TREG to suppress cancer depends on interleukin (IL)‐10, which serves to maintain immune homeostasis within bowel and support a protective antiinflammatory TREG phenotype. However, under proinflammatory conditions, TREG may fail to provide antiinflammatory protection and instead contribute to a T helper (Th)‐17‐driven procarcinogenic process; a cancer state that is reversible by downregulation of inflammation. Consequently, hygienic individuals with a weakened IL‐10 and TREG‐mediated inhibitory loop are highly susceptible to the carcinogenic consequences of elevated IL‐6 and IL‐17 and show more frequent inflammation‐associated cancers. Taken together, these data unify seemingly divergent disease processes such as autoimmunity and cancer and help explain the paradox of TREG and inflammation in cancer. Enhancing protective TREG functions may promote healthful longevity and significantly reduce risk of cancer.

Inflammation-induced cell proliferation potentiates DNA damage-induced mutations in vivo.

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence.

Age-dependent accumulation of recombinant cells in the mouse pancreas revealed by in situ fluorescence imaging.

Mitotic homologous recombination (HR) is critical for the repair of double-strand breaks, and conditions that stimulate HR are associated with an increased risk of deleterious sequence rearrangements that can promote cancer. Because of the difficulty of assessing HR in mammals, little is known about HR activity in mammalian tissues or about the effects of cancer risk factors on HR in vivo. To study HR in vivo, we have used fluorescent yellow direct repeat mice, in which an HR event at a transgene yields a fluorescent phenotype. Results show that HR is an active pathway in the pancreas throughout life, that HR is induced in vivo by exposure to a cancer chemotherapeutic agent, and that recombinant cells accumulate with age in pancreatic tissue. Furthermore, we developed an in situ imaging approach that reveals an increase in both the frequency and the sizes of isolated recombinant cell clusters with age, indicating that both de novo recombination events and clonal expansion contribute to the accumulation of recombinant cells with age. This work demonstrates that aging and exposure to a cancer chemotherapeutic agent increase the frequency of recombinant cells in the pancreas, and it also provides a rapid method for revealing additional factors that modulate HR and clonal expansion in vivo.

Integrated Molecular Analysis Indicates Undetectable Change in DNA Damage in Mice after Continuous Irradiation at ~ 400-Fold Natural Background Radiation

Background: In the event of a nuclear accident, people are exposed to elevated levels of continuous low dose-rate radiation. Nevertheless, most of the literature describes the biological effects of acute radiation. Objectives: DNA damage and mutations are well established for their carcinogenic effects. We assessed several key markers of DNA damage and DNA damage responses in mice exposed to low dose-rate radiation to reveal potential genotoxic effects associated with low dose-rate radiation. Methods: We studied low dose-rate radiation using a variable low dose-rate irradiator consisting of flood phantoms filled with 125Iodine-containing buffer. Mice were exposed to 0.0002 cGy/min (~ 400-fold background radiation) continuously over 5 weeks. We assessed base lesions, micronuclei, homologous recombination (HR; using fluorescent yellow direct repeat mice), and transcript levels for several radiation-sensitive genes. Results: We did not observe any changes in the levels of the DNA nucleobase damage products hypoxanthine, 8-oxo-7,8-dihydroguanine, 1,N6-ethenoadenine, or 3,N4-ethenocytosine above background levels under low dose-rate conditions. The micronucleus assay revealed no evidence that low dose-rate radiation induced DNA fragmentation, and there was no evidence of double strand break–induced HR. Furthermore, low dose-rate radiation did not induce Cdkn1a, Gadd45a, Mdm2, Atm, or Dbd2. Importantly, the same total dose, when delivered acutely, induced micronuclei and transcriptional responses. Conclusions: These results demonstrate in an in vivo animal model that lowering the dose-rate suppresses the potentially deleterious impact of radiation and calls attention to the need for a deeper understanding of the biological impact of low dose-rate radiation.

CD4+ lymphocytes modulate prostate cancer progression in mice

Chronic inflammation contributes to the development of prostate cancer in humans. Here, we show that male ApcMin/+ mice also develop prostate carcinoma with increasing age, mimicking that seen in humans in their 5th or 6th decade of life. Proinflammatory cytokines were significantly linked with cancer and increasing age in our mouse model; however, prostate and bowel tissues lacked evidence of inflammatory cell infiltrates other than mast cells. Lymphocytes protected against cancer, and protection from prostate cancer resided in antiinflammatory CD4+CD25+ regulatory (TREG) cells that downregulated inflammatory cytokines. Supplementation with syngeneic TREG cells collected from wild‐type mice reduced the levels of interleukin (IL)‐6 (p < 0.05) and IL‐9 (p < 0.001) and lowered prostate cancer risk (p < 0.05). Depletion of CD25+ cells in 2‐month‐old animals increased the expression of IL‐6 (p < 0.005) within prostate and increased the frequency of high‐grade prostatic intraepithelial neoplasia (p < 0.05) and microinvasive prostatic carcinoma (p < 0.05) in dorsolateral prostate. Depletion of CD25+ cells in young animals also increased the frequency of intestinal cancer in Min mice. Taken together, chronically elevated proinflammatory cytokines promoted carcinoma in ApcMin/+ mice. TREG lymphocytes downregulated inflammation‐associated carcinogenic processes and contributed to immune and epithelial homeostasis.

Applications of fluorescence for detecting rare sequence rearrangements in vivo.

Homologous recombination (HR) is an important pathway for the accurate repair of potentially cytotoxic or mutagenic double strand breaks (DSBs), as well as double strand ends that arise due to replication fork breakdown. Thus, measuring HR events can provide information on conditions that induce DSB formation and replicative stress. To study HR events in vivo, we previously developed Fluorescent Yellow Direct Repeat (FYDR) mice in which a recombination event at an integrated transgene yields a fluorescent signal. Recently, we published an application of these mice demonstrating that fluorescent recombinant cells can be directly detected within intact pancreatic tissue. Here, we show that in situ imaging is a more sensitive method for detecting exposure-induced recombinant cells, yielding statistical significance with smaller cohorts. In addition, we show inter-mouse and gender-dependent variation in transgene expression, examine its impact on data interpretation, and discuss solutions to overcoming the effects of such variation. Finally, we also present data on EYFP expression, showing that several tissues, in addition to the pancreas, may be amenable for in situ detection of recombinant cells in the FYDR mice. The FYDR mice provide a unique tool for identifying genetic conditions and environmental exposures that induce genotoxic stress in a variety of tissues.

Tissue-specific differences in the accumulation of sequence rearrangements with age.

Mitotic homologous recombination (HR) is a critical pathway for the accurate repair of DNA double strand breaks (DSBs) and broken replication forks. While generally error-free, HR can occur between misaligned sequences, resulting in deleterious sequence rearrangements that can contribute to cancer and aging. To learn more about the extent to which HR occurs in different tissues during the aging process, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which an HR event in a transgene yields a fluorescent phenotype. Here, we show tissue-specific differences in the accumulation of recombinant cells with age. Unlike pancreas, which shows a dramatic 23-fold increase in recombinant cell frequency with age, skin shows no increase in vivo. In vitro studies indicate that juvenile and aged primary fibroblasts are similarly able to undergo HR in response to endogenous and exogenous DNA damage. Therefore, the lack of recombinant cell accumulation in the skin is most likely not due to an inability to undergo de novo HR events. We propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of recombinant cells to persist and clonally expand within tissues.

Development and characterization of a novel variable low-dose rate irradiator for in vivo mouse studies

Radiation exposure of humans generally results in low doses delivered at low dose rate. Our limited knowledge of the biological effects of low dose radiation is mainly based on data from the atomic bomb Life Span Study (LSS) cohort. However, the total doses and dose rates in the LSS cohort are still higher than most environmental and occupational exposures in humans. Importantly, the dose rate is a critical determinant of health risks stemming from radiation exposure. Understanding the shape of the dose-rate response curve for different biological outcomes is thus crucial for projecting the biological hazard from radiation in different environmental and man-made conditions. A significant barrier to performing low dose-rate studies is the difficulty in creating radiation source configurations compatible with long-term cellular or animal experiments. In this study the design and characterization of a large area, 125I-based irradiator is described. The irradiator allows continuous long-term exposure of mice at variable dose rates and can be sited in standard animal care facilities. The dose rate is determined by the level of 125I activity added to a large NaOH-filled rectangular phantom. The desired dose rate is maintained at essentially constant levels by weekly additions of 125I to compensate for decay. Dosimetry results for long-term animal irradiation at targeted dose rates of 0.00021 and 0.0021 cGy min−1 are presented.