Low doses of ionizing radiation induce immune-stimulatory responses in isolated human primary monocytes

The health effects arising from exposure to low doses of ionizing radiation are of particular concern, mainly due to the increased application of diagnostic and therapeutic X-ray modalities. The mechanisms behind the cell and tissue responses to low doses remain to be elucidated. Accumulating evidence suggests that low doses of ionizing radiation induce activation of the immune response; however, the processes involved have yet to be adequately investigated. Monocytes are key players in the induction of an immune response. Within the context of this study, we investigated the activation of toll-like receptors (TLRs), mitogen-activated protein kinases (MAPKs) and NF-B signaling in isolated human primary monocytes in response to low doses (0.05 and 0.1 Gy) and a high dose (1 Gy) of ionizing radiation. Using quantitative RT-PCR and ELISA techniques, our results showed a positive regulation of TLR signaling in response to low doses but a less significant response at high doses. This activation was demonstrated via the activation of TLR signaling molecules (HMGB1, TLR4, TLR9, MyD88 and IRAK1). Furthermore, and in contrast to the high dose, the low doses showed increased phosphorylation levels of the protein IB, and therefore positive signaling of the NF-B pathway. This result denotes pro-survival and pro-inflammatory responses. Additionally, MAPKs were activated in response to 0.05 Gy, while 0.1 and 1 Gy showed a downregulatory trend that may be related to activation of the PF4 gene. On the other hand, there was highly significant involvement of activated p53 and damaged genes in response to high but not low doses. In conclusion, this study addressed the need to re-evaluate health risks arising from exposure to low doses of ionizing radiation, particularly in view of the accumulating evidence reporting inflammatory and oncogenic consequences from these exposures

Intensity modulated radiotherapy induces pro-inflammatory and pro-survival responses in prostate cancer patients

Intensity modulated radiotherapy (IMRT) is one of the modern conformal radiotherapies that is widely used within the context of cancer patient treatment. It uses multiple radiation beams targeted to the tumor, however, large volumes of the body receive low doses of irradiation. Using γ-H2AX and global genome expression analysis, we studied the biological responses induced by low doses of ionizing radiation in prostate cancer patients following IMRT. By means of different bioinformatics analyses, we report that IMRT induced an inflammatory response via the induction of viral, adaptive, and innate immune signaling. In response to growth factors and immune-stimulatory signaling, positive regulation in the progression of cell cycle and DNA replication were induced. This denotes pro-inflammatory and pro-survival responses. Furthermore, double strand DNA breaks were induced in every patient 30 min after the treatment and remaining DNA repair and damage signaling continued after 18-24 h. Nine genes belonging to inflammatory responses (TLR3, SH2D1A and IL18), cell cycle progression (ORC4, SMC2 and CCDC99) and DNA damage and repair (RAD17, SMC6 and MRE11A) were confirmed by quantitative RT-PCR. This study emphasizes that the risk assessment of health effects from the out-of-field low doses during IMRT should be of concern, as these may increase the risk of secondary cancers and/or systemic inflammation

Biological responses after low doses X-ray exposure: gene expression and mechanistic studies

The health effects arising from exposure to low doses of ionizing radiation are increasing due to the extensive use of medical and diagnostic applications. One of the central questions in the field of radiation biology is understanding the health consequences of these low doses, where several factors like genetics, life-style, and lack of robust epidemiological studies are impeding well-validated conclusions. Within the context of this PhD, we aim to understand the biological responses after low dose exposures in both in vitro and in vivo studies. The first part of this PhD is composed of five different chapters that aim to introduce the main objectives of our research and discuss the complexity of low dose research. Ionizing radiation contributes to the development in many of the sectors like medical diagnosis, therapy, industry, and agriculture. However, exposure to these radiations can carry serious health consequences, like cancer or non-cancer diseases. In Chapter 1, we give a short overview about the physical properties of ionizing radiation and basic concepts of dosimetry. Furthermore, we discuss the main sources of ionizing radiation and the epidemiological health studies that aimed to investigate the effects of ionizing radiation. Moreover, we discuss both deterministic and stochastic effects. On the other hand, we give a thorough overview about the risk assessment models used to estimate the risk after ionizing radiation exposures. In particular, we explain the linear-no threshold (LNT) hypothesis, which is based on linear extrapolation from high to low doses; however, several controversies challenge this hypothesis, mainly due to mechanisms involved in cellular communications involved in the low dose range. DNA double strand breaks may induce mutations and contribute to the development of cancers if misrepaired or unrepaired. In Chapter 2, we give a general overview about the cellular and molecular mechanisms involved in radiation responses. These include DNA damage signalling and repair. Furthermore, we discuss the different mechanisms involved in radiation-induced cell death, such as apoptosis and senescence. One of the challenging objectives within the context of low dose studies is the characterization of low dose specific biomarkers that are sensitive and robust. In Chapter 3, we highlight the basic considerations for choosing a biomarker for epidemiological studies. On the other hand, we discuss possible biomarkers that can be of use in the low dose range. DNA double strand break studies, via scoring of the Ser 139 phosphorylated form of the histone H2AX (γ-H2AX) is one of the most sensitive biomarkers for DNA damage and has been shown to be sensitive for low doses; but also, oxidative stress biomarkers must be investigated as well. Along with DNA damage studies, transcriptional and translational changes could provide "rich" information about the mechanisms involved in low dose responses, in particular whole genome analysis and cytokine measurement were shown to be reliable and sensitive techniques for radiation low dose research. Our research has shown that immune responses play a central role after low dose exposures. In Chapter 4, we give an overview about the different immune modulatory responses involved in radiation responses. First, we discuss the radiosensitivity of the different immune blood cells followed by an overview about the basic immune pathways, such as NF-κB, MAPK and toll-like receptor (TLR) signalling and their involvement in radiation responses. The introduction part is complemented with Chapter 5 that discusses the aims of the thesis and the outline of the research. The second part of this PhD is composed of three different scientific papers that discuss the results combined for this dissertation. To understand better the differences in responses between low and high doses of ionizing radiation, aliquots of whole blood samples were collected from ten healthy donors and irradiated in vitro with high and low doses of ionizing radiation. Using whole genome analysis and different bioinformatics approaches, we unravelled that low doses of ionizing radiation are characterized by the induction of an immune response by the activation of chemokine and cytokine signalling; furthermore, there they exhibit a pro-inflammatory response due to the activation of NF-κB, MAPK and TLR signalling. On other hand, the high dose response is characterized by damage signalling, mainly by the involvement of p53. Several genes belonging to immune and damaging responses were confirmed with quantitative RT-PCR. The results of this study are discussed in Chapter 6. In an aim to confirm our in vitro whole genome analysis, we investigated the response to low doses of ionizing radiation in vivo. Because samples collected from human would be more reliable than animal studies, we chose prostate cancer patients undergoing intensity modulated radiotherapy (IMRT) as target population. These patients are adequate for low dose studies because they receive a high dose to the tumour; however, large volumes of normal tissues receive low doses. In an attempt to unravel the biological responses, whole blood samples were collected before and after the first fraction of IMRT. DNA double strand breaks and whole genome analysis were performed. Using different bioinformatics analysis, we showed that the main response after low dose exposure is the induction of immune-related response, which is composed of DNA damage and inflammatory responses, growth factor signalling and positive regulation of cell cycle. Immune responses and growth factors were shown to be related to the positive regulation of cell cycle progression; this indicates that cell cycle arrest was not activated; nevertheless, DNA damage signalling was not completed. Using quantitative RT-PCR, various genes belonging to different biological pathways were confirmed. The results of this study are presented in Chapter 7. Monocytes are central players in the induction of immune response via the secretion of immune-stimulatory cytokines and the activation of immune-related pathways, mainly Toll-like receptors (TLRs), NF-κB and mitogen activated protein kinases (MAPKs). In an attempt to confirm the immune-stimulatory nature of the responses combined from both the in vitro and in vivo studies, we investigated the activation of immune pathways in monocytes isolated from human donors. Our results showed that low doses positively regulate the TLR, NF-κB and MAPKs signalling; however high doses did show less involvement of these pathways, which might suggest immune-suppressive responses. The results of this study are presented in Chapter 8. The third part of this PhD thesis comprises the general discussion. It is composed of two different chapters that intend to explain all the results combined from the different studies with conclusions and perspectives. Taking into account the results presented in Chapters 6, 7, and 8, we provide in Chapter 9 a thorough explanation about the differences between responses after low and high dose exposures. These are divided into immune responses, growth factor signalling, and damage responses. Furthermore, we discuss the involvement of the different pathways, namely MAPKs, NF-κB and growth factors in cancer development and inflammation. On the other hand, we discuss the role of NF-κB, MAPKs and growth factors as demonstrated bystander effects players. Finally, we give an overview about the different bioinformatics approaches that could be useful for whole genome analysis of low dose responses used in our studies as well as those described in the literature. In addition to that, in Chapter 10, we conclude that low and high doses of ionizing radiation are characterized by different responses, thus extrapolation is not an accurate approach. Furthermore, we suggest that radiation protection measures and dose reducing techniques should be applied in the medical field. On the other hand, and in an attempt to develop our understanding to low dose responses, we recommend performing mechanistic studies and molecular epidemiology for clear-cut answers concerning low dose health effects. However, we discuss the many challenges that hinder the fast progression of low dose research, mainly because there is a difficulty in choosing a model for biological studies

Gene set enrichment analysis highlights different gene expression profiles in whole blood samples X-irradiated with low and high doses

Purpose : Health risks from exposure to low doses of ionizing radiation (IR) are becoming a concern due to the rapidly growing medical applications of X-rays. Using microarray techniques, this study aims for a better understanding of whole blood response to low and high doses of IR. Materials and methods : Aliquots of peripheral blood samples were irradiated with 0, 0.05, and 1 Gy X-rays. RNA was isolated and prepared for microarray gene expression experiments. Bioinformatic approaches, i.e., univariate statistics and Gene Set Enrichment Analysis (GSEA) were used for analyzing the data generated. Seven differentially expressed genes were selected for further confirmation using quantitative real-time PCR (RT-PCR). Results : Functional analysis of genes differentially expressed at 0.05 Gy showed the enrichment of chemokine and cytokine signaling. However, responsive genes to 1 Gy were mainly involved in tumor suppressor protein 53 (p53) pathways. In a second approach, GSEA showed a higher statistical ranking of inflammatory and immune-related gene sets that are involved in both responding and/or secretion of growth factors, chemokines, and cytokines. This indicates the activation of the immune response. Whereas, gene sets enriched at 1 Gy were 'classical' radiation pathways like p53 signaling, apoptosis, DNA damage and repair. Comparative RT-PCR studies showed the significant induction of chemokine-related genes (PF4, GNG11 and CCR4) at 0.05 Gy and DNA damage and repair genes at 1 Gy (DDB2, AEN and CDKN1A). Conclusions : This study moves a step forward in understanding the different cellular responses to low and high doses of X-rays. In addition to that, and in a broader context, it addresses the need for more attention to the risk assessment of health effects resulting from the exposure to low doses of IR

Tight Junction Proteins and the Biology of Hepatobiliary Disease.

Tight junctions (TJ) are intercellular adhesion complexes on epithelial cells and composed of integral membrane proteins as well as cytosolic adaptor proteins. Tight junction proteins have been recognized to play a key role in health and disease. In the liver, TJ proteins have several functions: they contribute as gatekeepers for paracellular diffusion between adherent hepatocytes or cholangiocytes to shape the blood-biliary barrier (BBIB) and maintain tissue homeostasis. At non-junctional localizations, TJ proteins are involved in key regulatory cell functions such as differentiation, proliferation, and migration by recruiting signaling proteins in response to extracellular stimuli. Moreover, TJ proteins are hepatocyte entry factors for the hepatitis C virus (HCV)-a major cause of liver disease and cancer worldwide. Perturbation of TJ protein expression has been reported in chronic HCV infection, cholestatic liver diseases as well as hepatobiliary carcinoma. Here we review the physiological function of TJ proteins in the liver and their implications in hepatobiliary diseases.journal articlereview2020 Jan 282020 01 28importe

EPI-CT: in vitro assessment of the applicability of the γ-H2AX-foci assay as cellular biomarker for exposure in a multicentre study of children in diagnostic radiology

Purpose : To conduct a feasibility study on the application of the gamma-H2AX foci assay as an exposure biomarker in a prospective multicentre paediatric radiology setting. Materials and methods : A set of in vitro experiments was performed to evaluate technical hurdles related to biological sample collection in a paediatric radiology setting (small blood sample volume), processing and storing of blood samples (effect of storing blood at 4 degrees C), the reliability of foci scoring for low-doses (merge gamma-H2AX/53BP1 scoring), as well as the impact of contrast agent administration as potential confounding factor. Given the exploratory nature of this study and the ethical constraints related to paediatric blood sampling, blood samples from adult volunteers were used for these experiments. In order to test the feasibility of pooling the gamma-H2AX data when different centres are involved in an international multicentre study, two intercomparison studies in the low-dose range (10-500 mGy) were performed. Results : Determination of the number of X-ray induced gamma-H2AX foci is feasible with one 2 ml blood sample pre- and post-computed tomography (CT) scan. Lymphocyte isolation and fixation on slides is necessary within 5 h of blood sampling to guarantee reliable results. The possible enhancement effect of contrast medium on the induction of DNA DSB in a patient study can be ruled out if radiation doses and the contrast agent concentration are within diagnostic ranges. The intercomparison studies using in vitro irradiated blood samples showed that the participating laboratories, executing successfully the gamma-H2AX foci assay in lymphocytes, were able to rank blind samples in order of lowest to highest radiation dose based on mean foci/cell counts. The dose response of all intercomparison data shows that a dose point of 10 mGy could be distinguished from the sham-irradiated control (p = 0.006). Conclusions : The results demonstrate that it is feasible to apply the gamma-H2AX foci assay as a cellular biomarker of exposure in a multicentre prospective study in paediatric CT imaging after validating it in an in vivo international pilot study on paediatric patients

Hepatitis B virus compartmentalization and single-cell differentiation in hepatocellular carcinoma

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) world-wide. The molecular mechanisms of viral hepatocarcinogenesis are still partially understood. Here, we applied two complementary single-cell RNA-sequencing protocols to investigate HBV-HCC host cell interactions at the single cell level of patient-derived HCC. Computational analyses revealed a marked HCC heterogeneity with a robust and significant correlation between HBV reads and cancer cell differentiation. Viral reads significantly correlated with the expression of HBVdependency factors such as HLF in different tumor compartments. Analyses of virus-induced host responses identified previously undiscovered pathways mediating viral carcinogenesis, such as E2F- and MYC targets as well as adipogenesis. Mapping of fused HBV-host cell transcripts allowed the characterization of integration sites in individual cancer cells. Collectively, single-cell RNASeq unravels heterogeneity and compartmentalization of both, virus and cancer identifying new candidate pathways for viral hepatocarcinogenesis. The perturbation of pro-carcinogenic gene expression even at low HBV levels highlights the need of HBV cure to eliminate HCC risk

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets

International audienceOBJECTIVE:Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent.DESIGN:Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection.RESULTS:Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets.CONCLUSION:The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure

Interleukin-32 Contributes to Human Nonalcoholic Fatty Liver Disease and Insulin Resistance

PMC6719754Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder due to increased accumulation of fat in the liver and in many cases to enhanced inflammation. Although the contribution of inflammation in the pathogenesis of NAFLD is well established, the cytokines that are involved and how they influence liver transformation are still poorly characterized. In addition, with other modifiers, inflammation influences NAFLD progression to liver cirrhosis and hepatocellular carcinoma, demonstrating the need to find new molecular targets with potential future therapeutic applications. We investigated gene signatures in 38 liver biopsies from patients with NAFLD and obesity who had received bariatric surgery and compared these to 10 control patients who had received a cholecystectomy, using DNA microarray technology. A subset of differentially expressed genes was then validated on a larger cohort of 103 patients who had received bariatric surgery for obesity; data were thoroughly analyzed in terms of correlations with NAFLD pathophysiological parameters. Finally, the impact of a specific cytokine, interleukin-32 (IL32), was addressed on primary human hepatocytes (PHHs). Transcript analysis revealed an up-regulation of proinflammatory cytokines IL32, chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL10 and of ubiquitin D (UBD), whereas down-regulation of insulin-like growth factor-binding protein 2 (IGFBP2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was reported in patients with NAFLD. Moreover, IL32, which is the major deregulated gene, correlated with body mass index (BMI), waist circumference, NAFLD activity score (NAS), aminotransferases (alanine aminotransferase [ALAT] and aspartate aminotransferase [ASAT]), and homeostasis model assessment of insulin resistance (HOMA-IR) index in patients. Consistent with an instrumental role in the pathophysiology of NAFLD, treatment of control human hepatocytes with recombinant IL32 leads to insulin resistance, a hallmark metabolic deregulation in NAFLD hepatocytes. Conclusion: IL32 has a critical role in the pathogenesis of NAFLD and could be considered as a therapeutic target in patients