Christine Linard

Gene Expression Profile in Human Late Radiation Enteritis Obtained by High-Density cDNA Array Hybridization

Abstract Vozenin-Brotons, M-C., Milliat, F., Linard, C., Strup, C., François, A., Sabourin, J-C., Lasser, P., Lusinchi, A., Deutsch, E., Girinsky, T., Aigueperse, J., Bourhis, J. and Mathé, D. Gene Expression Profile in Human Late Radiation Enteritis Obtained by High-Density cDNA Array Hybridization. Radiat. Res. 161, 299–311 (2004). Late radiation enteritis is a sequela of radiation therapy to the abdomen. The pathogenic process is poorly understood at the molecular level. cDNA array analysis was used to provide new insights into the pathogenesis of this disorder. Gene profiles of six samples of fibrotic bowel tissue from patients with radiation enteritis and six healthy bowel tissue samples from patients without radiation enteritis were compared using membrane-based arrays containing 1314 cDNAs. Results were confirmed with real-time RT-PCR and Western blot analysis. Array analysis identified many differentially expressed genes involved in fibrosis, stress response, inflammation, cell adhesion, intracellular and nuclear signaling, and metabolic pathways. Increased expression of genes coding for proteins involved in the composition and remodeling of the extracellular matrix, along with altered expression of genes involved in cell- to-cell and cell-to-matrix interactions, were observed mainly in radiation enteritis samples. Stress, inflammatory responses, and antioxidant metabolism were altered in radiation enteritis as were genes coding for recruitment of lymphocytes and macrophages. The Rho/HSP27 (HSPB1)/zyxin pathway, involved in tissue contraction and myofibroblast transdifferentiation, was also altered in radiation enteritis, suggesting that this pathway could be related to the fibrogenic process. Our results provide a global and integrated view of the alteration of gene expression associated with radiation enteritis. They suggest that radiation enteritis is a dynamic process involving constant remodeling of each structural component of the intestinal tissue, i.e. the mucosa, the mesenchyme, and blood vessels. Functional studies will be necessary to validate the present results.

Abdominal γ-Radiation Induces an Accumulation of Function-Impaired Regulatory T Cells in the Small Intestine

PURPOSE To assess the frequency and the functional characteristics of one major component of immune tolerance, the CD4(+)FoxP3(+) regulatory T cells (Tregs) in a mouse model of abdominal irradiation. METHODS AND MATERIALS Mice were exposed to a single abdominal dose of γ-radiation (10 Gy). We evaluated small intestine Treg infiltration by Foxp3 immunostaining and the functional suppressive activity of Tregs isolated from mesenteric lymph nodes. RESULTS Foxp3 immunostaining showed that radiation induced a long-term infiltration of the intestine by Tregs (levels 5.5 times greater than in controls). Co-culture of Tregs from mesenteric lymph nodes with CD4(+) effector cells showed that the Tregs had lost their suppressive function. This loss was associated with a significant decrease in the levels of Foxp3, TGF-β, and CTLA-4 mRNA, all required for optimal Treg function. At Day 90 after irradiation, Tregs regained their suppressive activity as forkhead box P3 (Foxp3), transforming growth factor beta (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression returned to normal. Analysis of the secretory function of mesenteric lymph node Tregs, activated in vitro with anti-CD3/anti-CD28 Abs, showed that this dysfunction was independent of a defect in interleukin-10 secretion. CONCLUSION Radiation caused a long-term accumulation of function-impaired Foxp3(+)CD4(+) Tregs in the intestine. Our study provides new insights into how radiation affects the immune tolerance in peripheral tissues.

Reduction of PPARγ Expression by γ-irradiation as a Mechanism Contributing to Inflammatory Response in Rat Colon: Modulation by the 5-ASA Agonist

Radiation-induced intestinal injuries, including inflammation and immune response, remain a limiting factor in the effectiveness of pelvic radiotherapy and in the patients quality of life during and after treatment. Peroxisome proliferation-activated receptor (PPAR) agonists are now emerging as therapeutic drugs for various inflammatory diseases that are characterized by impaired PPAR expression. The purpose of this study was to investigate the profile of PPAR expression in rat colonic mucosa 3 and 7 days after abdominal γ-irradiation (10 Gy). We tested whether irradiation-induced acute inflammatory response could be modulated pharmacologically with the antiinflammatory properties of 5-aminosalicylic acid (5-ASA) (250 mg/kg/day), which is a PPAR activator. Irradiation drastically reduced mRNA and protein levels of PPARα and -γ and of the heterodimer retinoid X receptor (RXR)α at 3 days postirradiation. 5-ASA treatment normalized both PPARγ and RXRα expression at 3 days postirradiation and PPARα at 7 days. By promoting PPAR expression and its nuclear translocation, 5-ASA interfered with the nuclear factor (NF)-κB pathway, both by reducing irradiation-induced NF-κB p65 translocation/activation and increasing the expression of nuclear factor-κB inhibitor (IκB) mRNA and protein. Therefore, 5-ASA prevents irradiation-induced inflammatory processes as well as expression of tumor necrosis factor α, monocyte chemotactic protein-1, inducible nitric-oxide synthase, and macrophage infiltration. In addition, 5-ASA restores the interferon γ/signal transducer and activator of transcription (STAT)-1 and STAT-3 concentrations that were impaired at 3 and 7 days postirradiation and are correlated with suppressor of cytokine signaling-3 repression. Collectively, these results indicate that PPAR agonists may be effective in the prevention of inflammatory processes and immune responses during and after pelvic radiotherapy.

Modulation of gut substance P after whole-body irradiation : A new pathological feature

Exposure to ionizing radiation induces gastrointestinal dysfunction and inflammatory reactions. The present study carried out in the rat, focuses on substance P, an inflammatory mediator implicated in the control of intestinal motility. We have investigated the effects of gamma irradiation on plasma and tissue substance P levels, ileal smooth muscle activity, and properties of specific receptors. Plasma and ileal (mucosa and muscle) substance P concentrations were measured by radioimmunoassay. At doses ranging from 1 to 8 Gy, plasma substance P levels increased in a dose-dependent manner up to four days after irradiation. Ileal mucosal concentration decreased rapidly 1 hr after a 6-Gy irradiation as compared to controls. A second class of binding sites appeared three days after 6 Gy irradiation. In addition, substance P contractile effects measured on isolated ileum showed a fourfold decrease of EC50, three days after 6 Gy irradiation. These results indicated that gamma irradiation induced an increase of plasma levels concomitant with a modification of gastrointestinal substance P specific binding sites and contractile activity.

Intestinal irradiation and fibrosis in a Th1-deficient environment.

PURPOSE Changes in the Th1/Th2 immune balance may play a role in increasing the incidence of radiation-induced toxicity. This study evaluates the consequences of Th1 deficiency on intestinal response (fibrosis and T cell trafficking) to abdominal irradiation and examines in mucosa and mesenteric lymph nodes (MLN) the differential involvement of the two Th1 pathways, T-bet/STAT1 and IL-12/STAT4, in controlling this balance in mice. METHODS AND MATERIALS Using T-bet-deficient mice (T-bet-/-), we evaluated the mRNA and protein expression of the Th1 pathways (IFN-γ, T-bet/STAT1, and IL-12/STAT4) and the CD4+ and CD8+ populations in ileal mucosa and MLN during the first 3 months after 10 Gy abdominal irradiation. RESULTS The T-bet-deficient mice showed an increased fibrotic response to radiation, characterized by higher TGF-β1, col3a1 expression, and collagen deposition in mucosa compared with wild-type mice. This response was associated with drastically lower expression of IFN-γ, the hallmark Th1 cytokine. Analysis of the Th1 expression pathways, T-bet/STAT1 and IL-12/STAT4, showed their equal involvement in the failure of Th1 polarization. A minimal IFN-γ level depended on the IL-23-p19/STAT4 level. In addition, the radiation-induced deficiency in the priming of Th1 by IFN-γ was related to the defective homing capacity of CD8+ cells in the mucosa. CONCLUSION Irradiation induces Th2 polarization, and the Th2 immune response may play a role in potentiating irradiation-induced intestinal collagen deposition.

Involvement of primary afferent nerves after abdominal irradiation: consequences on ileal contractile activity and inflammatory mediator release in the rat.

In this study we analyzed the role of substance P (SP) from afferent nerves in ileum contractibility and in the release of inflammatory mediators (neurotensin, Il-1β, and TNF-α) in ileal mucosa and muscularis layers after a 10-Gy γ-irradiation of the abdomen. Six hours after irradiation, SP concentrations were lower than in control rats, and 3 days after irradiation SP-induced contractile activity was higher. Irradiation significantly increased the levels of neurotensin, Il-1β, and TNF-α in both layers. Pretreatment with capsaicin depleted afferent nerve endings of SP and reduced SP levels by about 50%. Capsaicin treatment reduced SP concentrations further, beyond the levels due to irradiation, thereby suggesting that all sources of SP are affected by irradiation. Capsaicin treatment prevented the irradiation from affecting SP-induced contractile response or increasing neurotensin levels. This finding suggests that SP released by afferent nerve endings controls these functions. Proinflammatory cytokine release was not reduced by capsaicin treatment.

Collapse of Skin Antioxidant Status during the Subacute Period of Cutaneous Radiation Syndrome: A Case Report

Abstract Benderitter, M., Isoir, M., Buard, V., Durand, V., Linard, C., Vozenin-Brotons, M. C., Steffanazi, J., Carsin, H. and Gourmelon, P. Collapse of Skin Antioxidant Status during the Subacute Period of Cutaneous Radiation Syndrome: A Case Report. Radiat. Res. 167, 43–50 (2007). This case report describes a patient suffering from accidental cutaneous radiation syndrome. Clinical symptoms were characterized by the presence of moist epidermal denudation over approximately 8% of the body surface without signs of necrosis 88 days after radiation exposure. The skin transcriptional profile was obtained and provides a comprehensive overview of the changes in gene expression associated with skin wound healing after irradiation. In particular, our data show a specific set of genes, i.e. SOD1, GPX1, TDX1, TDX2 and HSP60, implicated in the redox control of normal skin repair after radiation exposure, whereas HOX1 and HOX2 were involved in the pathological skin repair. A reduction in the antioxidant capacity of the irradiated tissue concomitant with a progressive establishment of an uncontrolled inflammatory response was noted. Our data corroborate the hypothesis that ROS modulation is a key element of the healing response after cutaneous exposure to radiation and that the collapse of skin antioxidant status interferes directly with wound healing in skin after radiation exposure. Thus a better understanding of the molecular events through which oxidative stress modulates the healing response could result in a more rational therapeutic approach to the pathological process induced after exposure of skin to radiation.

Flagellin or Lipopolysaccharide Treatment Modified Macrophage Populations after Colorectal Radiation of Rats

Radiation-induced acute intestinal toxicity remains a major limitation to the delivery of tumoricidal doses of colorectal irradiation. Recent reports indicate that Toll-like receptor (TLR) agonists TLR4 and TLR5 protect against toxicity due to intestinal irradiation. The phenotype (M1 or M2) of macrophages expressing TLRs may play a role in tissue repair. The aim was to investigate whether administration of TLR4 agonist lipopolysaccharide (LPS) or TLR5 agonist flagellin after irradiation modified the recruitment and phenotype of colonic macrophages and improved tissue damage. Rats were exposed to single 20- or 27-Gy doses of colorectal irradiation. TLR4 agonist LPS or TLR5 agonist flagellin (at 50 or 200 µg/rat) was administered i.p. 3 days after irradiation. Flow cytometric analysis, immunostaining, and real-time polymerase chain reaction analysis were used to assess the M1/M2 phenotype and crypt cell proliferation 7 days after irradiation. Irradiation (20 and 27 Gy) increased TLR4+ and TLR5+ macrophage frequency in the mucosa. LPS or flagellin administration maintained this elevated frequency after the 27-Gy irradiation. LPS and flagellin drove macrophages toward the anti-inflammatory M2 phenotype by increasing Arg1 and CD163 expression and microenvironmental effector molecules (C-C motif chemokine 22, transforming growth factor-β1, and interleukin-10). Proliferating cell nuclear antigen immunostaining, Ki67 expression, and antimicrobial factor Reg3γ showed that the M2 shift correlated with epithelial regeneration. In conclusion, administration of either LPS or flagellin after colorectal irradiation may provide effective protection against epithelial remodeling. This tissue repair was associated with an M2 macrophage shift. Using TLR agonists to moderately activate innate immunity should be considered as a strategy for protecting healthy tissue from irradiation.

Acute ileal inflammatory cytokine response induced by irradiation is modulated by subdiaphragmatic vagotomy

Neural involvement plays a role in the genesis of the peripheral inflammatory process that contributes to the irradiation intestinal disorders. However, little is known about the role of vagus nerve in modulating inflammatory process in rat. Here, we have shown that the NF-kappaB activation was consistent with the acute overexpression of pro-inflammatory cytokines (IL- 1beta, TNF-alpha, IL-6) at 3, 6, and 12 h induced by whole-body irradiation (8 Gy). Subdiaphragmatic vagotomy reduced NF-kappaB activation and cytokine transcription in the early period post-irradiation. In contrast, vagotomy amplified overexpression of irradiation-induced anti-cytokines (IL-10, IL-1Ra) and of receptors involved in anti-inflammatory effects (IL- 1RII, TNFRII). These results show that the vagus nerve is a pro-inflammatory pathway in early irradiation-induced intestinal inflammation.

Flagellin and LPS each restores rat lymphocyte populations after colorectal irradiation

Radiation‐induced gastrointestinal toxicity, including its shift of the immune balance, remains a major limitation to delivering tumoricidal doses of abdominal radiation therapy. This study evaluates the effect on the colonˈs innate and adaptive immune responses to moderate irradiation doses and the therapeutic possibilities of maintaining immune homeostasis. We investigated whether administration of the TLR4 agonist LPS or of the TLR5 agonist flagellin, 3 days after a single 20‐Gy colorectal irradiation, modified recruitment of neutrophils, NK cells, or CD4+ or CD8+ T cells, 7 days postirradiation. Flow cytometric analysis showed that LPS and flagellin reduced irradiation‐induced neutrophil infiltration and normalized NK frequency. LPS normalized the CD4+ population and enhanced the CD8+ population, whereas flagellin maintained the radiation‐induced elevation in the frequencies of both. Irradiation also modified TLR4 and TLR5 expression on the surface of both populations, but LPS and flagellin each subsequently normalized them. LPS and flagellin were strong inducers of Th1 cytokines (IL‐12p35, IL‐12p40, and IFN‐γ) and thus, contributed to a shift from the Th2 polarization induced by irradiation toward a Th1 polarization, confirmed by an increase of the T‐bet:GATA3 ratio, which assesses the Th1 or Th2 status in mixed cell populations. LPS and flagellin treatment resulted in overexpression of FoxP3, IL‐2Rα (CD25), IL‐2, and OX40, all expressed specifically and involved in high levels of Treg cell expansion. We observed no variation in Treg function‐related expression of IL‐10 or CTLA‐4. These data suggest that the use of TLR ligands limits the effects of irradiation on innate and adaptive immunity.