HGF and TSG-6 Released by Mesenchymal Stem Cells Attenuate Colon Radiation-Induced Fibrosis.

Fibrosis is a leading cause of death in occidental states. The increasing number of patients with fibrosis requires innovative approaches. Despite the proven beneficial effects of mesenchymal stem cell (MSC) therapy on fibrosis, there is little evidence of their anti-fibrotic effects in colorectal fibrosis. The ability of MSCs to reduce radiation-induced colorectal fibrosis has been studied in vivo in Sprague-Dawley rats. After local radiation exposure, rats were injected with MSCs before an initiation of fibrosis. MSCs mediated a downregulation of fibrogenesis by a control of extra cellular matrix (ECM) turnover. For a better understanding of the mechanisms, we used an in vitro model of irradiated cocultured colorectal fibrosis in the presence of human MSCs. Pro-fibrotic cells in the colon are mainly intestinal fibroblasts and smooth muscle cells. Intestinal fibroblasts and smooth muscle cells were irradiated and cocultured in the presence of unirradiated MSCs. MSCs mediated a decrease in profibrotic gene expression and proteins secretion. Silencing hepatocyte growth factor (HGF) and tumor necrosis factor-stimulated gene 6 (TSG-6) in MSCs confirmed the complementary effects of these two genes. HGF and TSG-6 limited the progression of fibrosis by reducing activation of the smooth muscle cells and myofibroblast. To settle in vivo the contribution of HGF and TSG-6 in MSC-antifibrotic effects, rats were treated with MSCs silenced for HGF or TSG-6. HGF and TSG-6 silencing in transplanted MSCs resulted in a significant increase in ECM deposition in colon. These results emphasize the potential of MSCs to influence the pathophysiology of fibrosis-related diseases, which represent a challenging area for innovative treatments.

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-ß1, 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.

Systemically delivered adipose stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome by immunomodulating the inflammatory response through a CD11b+ cell-dependent mechanism.

BACKGROUND: Stromal vascular fraction (SVF) treatment promoted the regeneration of the intestinal epithelium, limiting lethality in a mouse model of radiation-induced gastrointestinal syndrome (GIS). The SVF has a heterogeneous cell composition; the effects between SVF and the host intestinal immunity are still unknown. The specific role of the different cells contained in the SVF needs to be clarified. Monocytes-macrophages have a crucial role in repair and monocyte recruitment and activation are orchestrated by the chemokine receptors CX3CR1 and CCR2. METHODS: Mice exposed to abdominal radiation (18 Gy) received a single intravenous injection of SVF (2.5 × 106 cells), obtained by enzymatic digestion of inguinal fat tissue, on the day of irradiation. Intestinal immunity and regeneration were evaluated by flow cytometry, RT-PCR and histological analyses. RESULTS: Using flow cytometry, we showed that SVF treatment modulated intestinal monocyte differentiation at 7 days post-irradiation by very early increasing the CD11b+Ly6C+CCR2+ population in the intestine ileal mucosa and accelerating the phenotype modification to acquire CX3CR1 in order to finally restore the F4/80+CX3CR1+ macrophage population. In CX3CR1-depleted mice, SVF treatment fails to mature the Ly6C-MCHII+CX3CR1+ population, leading to a macrophage population deficit associated with proinflammatory environment maintenance and defective intestinal repair; this impaired SVF efficiency on survival. Consistent with a CD11b+ being involved in SVF-induced intestinal repair, we showed that SVF-depleted CD11b+ treatment impaired F4/80+CX3CR1+macrophage pool restoration and caused loss of anti-inflammatory properties, abrogating stem cell compartment repair and survival. CONCLUSIONS: These data showed that SVF treatment mitigates the GIS-involving immunomodulatory effect. Cooperation between the monocyte in SVF and the host monocyte defining the therapeutic properties of the SVF is necessary to guarantee the effective action of the SVF on the GIS.

Fetal Muse-based therapy prevents lethal radio-induced gastrointestinal syndrome by intestinal regeneration.

BACKGROUND: Human multilineage-differentiating stress enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be easily obtained from various adult or fetal tissues. Regenerative effects of Muse cells have been shown in some disease models. Muse cells specifically home in damaged tissues where they exert pleiotropic effects. Exposition of the small intestine to high doses of irradiation (IR) delivered after radiotherapy or nuclear accident results in a lethal gastrointestinal syndrome (GIS) characterized by acute loss of intestinal stem cells, impaired epithelial regeneration and subsequent loss of the mucosal barrier resulting in sepsis and death. To date, there is no effective medical treatment for GIS. Here, we investigate whether Muse cells can prevent lethal GIS and study how they act on intestinal stem cell microenvironment to promote intestinal regeneration. METHODS: Human Muse cells from Wharton's jelly matrix of umbilical cord (WJ-Muse) were sorted by flow cytometry using the SSEA-3 marker, characterized and compared to bone-marrow derived Muse cells (BM-Muse). Under gas anesthesia, GIS mice were treated or not through an intravenous retro-orbital injection of 50,000 WJ-Muse, freshly isolated or cryopreserved, shortly after an 18 Gy-abdominal IR. No immunosuppressant was delivered to the mice. Mice were euthanized either 24 h post-IR to assess early small intestine tissue response, or 7 days post-IR to assess any regenerative response. Mouse survival, histological stainings, apoptosis and cell proliferation were studied and measurement of cytokines, recruitment of immune cells and barrier functional assay were performed. RESULTS: Injection of WJ-Muse shortly after abdominal IR highly improved mouse survival as a result of a rapid regeneration of intestinal epithelium with the rescue of the impaired epithelial barrier. In small intestine of Muse-treated mice, an early enhanced secretion of IL-6 and MCP-1 cytokines was observed associated with (1) recruitment of monocytes/M2-like macrophages and (2) proliferation of Paneth cells through activation of the IL-6/Stat3 pathway. CONCLUSION: Our findings indicate that a single injection of a small quantity of WJ-Muse may be a new and easy therapeutic strategy for treating lethal GIS.

Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells.

Hematopoietic stem cells (HSCs) are the rare cells responsible for the lifelong curative effects of hematopoietic cell (HC) transplantation. The demand for clinical-grade HSCs has increased significantly in recent decades, leading to major difficulties in treating patients. A promising but not yet achieved goal is the generation of HSCs from pluripotent stem cells. Here, we have obtained vector- and stroma-free transplantable HSCs by differentiating human induced pluripotent stem cells (hiPSCs) using an original one-step culture system. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multilineage hematopoietic population that can be serially transplanted. Single-cell RNA sequencing shows that this repopulating activity is due to a hematopoietic population that is transcriptionally similar to human embryonic aorta-derived HSCs. Overall, our results demonstrate the generation of HSCs from hiPSCs and will help identify key regulators of HSC production during human ontogeny.

The stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome in mice.

BACKGROUND: The intestine is particularly sensitive to moderate-high radiation dose and the development of gastrointestinal syndrome (GIS) leads to the rapid loss of intestinal mucosal integrity, resulting in bacterial infiltration, sepsis that comprise patient survival. There is an urgent need for effective and rapid therapeutic countermeasures. The stromal vascular fraction (SVF) derived from adipose tissue is an easily accessible source of cells with angiogenic, anti-inflammatory and regenerative properties. We studied the therapeutic impact of SVF and its action on the intestinal stem cell compartment. METHODS: Mice exposed to the abdominal radiation (18 Gy) received a single intravenous injection of stromal vascular fraction (SVF) (2.5 × 106 cells), obtained by enzymatic digestion of inguinal fat tissue, on the day of irradiation. Mortality was evaluated as well as intestinal regeneration by histological analyses and absorption function. RESULTS: The SVF treatment limited the weight loss of the mice and inhibited the intestinal permeability and mortality after abdominal irradiation. Histological analyses showed that SVF treatment stimulated the regeneration of the epithelium by promoting numerous enlarged hyperproliferative zones. SVF restored CD24+/lysozyme- and Paneth cell populations in the ISC compartment with the presence of Paneth Ki67+ cells. SVF has an anti-inflammatory effect by repressing pro-inflammatory cytokines, increasing M2 macrophages in the ileum and anti-inflammatory monocyte subtypes CD11b+Ly6clowCX3CR1high in the spleen. CONCLUSIONS: Through the pleiotropic effects that contribute to limiting radiation-induced lethality, SVF opens up attractive prospects for the treatment of emergency GIS.

Reduction of peroxisome proliferation-activated receptor gamma expression by gamma-irradiation as a mechanism contributing to inflammatory response in rat colon: modulation by the 5-aminosalicylic acid agonist.

Radiation-induced intestinal injuries, including inflammation and immune response, remain a limiting factor in the effectiveness of pelvic radiotherapy and in the patient's 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 gamma-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 PPARalpha and -gamma and of the heterodimer retinoid X receptor (RXR)alpha at 3 days postirradiation. 5-ASA treatment normalized both PPARgamma and RXRalpha expression at 3 days postirradiation and PPARalpha at 7 days. By promoting PPAR expression and its nuclear translocation, 5-ASA interfered with the nuclear factor (NF)-kappaB pathway, both by reducing irradiation-induced NF-kappaB p65 translocation/activation and increasing the expression of nuclear factor-kappaB inhibitor (IkappaB) mRNA and protein. Therefore, 5-ASA prevents irradiation-induced inflammatory processes as well as expression of tumor necrosis factor alpha, monocyte chemotactic protein-1, inducible nitric-oxide synthase, and macrophage infiltration. In addition, 5-ASA restores the interferon gamma/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.

Acute and persisting Th2-like immune response after fractionated colorectal gamma-irradiation.

AIM: To investigate if an immune imbalance may account for the development and progression of chronic radiation enteritis. We analyzed the Th1/Th2 immune response profile early and 6 mo after fractionated colorectal irradiation. METHODS: A rat model of fractionated colorectal gamma-irradiation (4-Gy fractions, 3 fractions per week) was designed to investigate the effects of cumulative dose on inflammatory mediators (cytokines and chemokines) and immune response (Th1/Th2 profile and immunosuppressive mediator IL-10) during acute (early) response and 6 mo after the end of fractionated irradiation (chronic response). Analyses were performed 1 d after the cumulative doses of 16 Gy and 36 Gy and 1 d, 3 d, and 26 wk after the cumulative dose of 52 Gy. RESULTS: Without causing histological damage, fractionated radiation induced elevated expression of IL-1beta, TNFalpha, MCP-1, and iNOS in distal colonic mucosa during the early post-irradiation phase. At that time, a Th2 profile was confirmed by expression of both the Th2-specific transcription factor GATA-3 and the chemokine receptor CCR4 and by suppression of the Th1 cytokine IFNgamma/IP-10 throughout the irradiation protocol. After 6 mo, despite the 2-fold reduction of iNOS and MCP-1 levels, the Th2 profile persisted, as shown by a 50% reduction in the expression of the Th1 transcription factor T-bet, the chemokine receptor CCXCR3, and the IFNgamma/STAT1 pathway. At the same time-point, the immunosuppressive IL-10/STAT3 pathway, known to regulate the Th1/Th2 balance, was expressed, in irradiated rats, at approximately half its level as compared to controls. This suppression was associated with an overexpression of SOCS3, which inhibits the feedback of the Th1 polarization and regulates IL-10 production. CONCLUSION: Colorectal irradiation induces Th2 polarization, defective IL-10/STAT3 pathway activation and SOCS3 overexpression. These changes, in turn, maintain a immunological imbalance that persists in the long term.

Long-term effectiveness of local BM-MSCs for skeletal muscle regeneration: a proof of concept obtained on a pig model of severe radiation burn.

BACKGROUND: Medical management of the severe musculocutaneous radiation syndrome involves surgical intervention with debridement of necrotic tissue. Even when skin excision is replaced by specific plastic surgery, treatment of the muscle radiation injury nonetheless remains difficult, for it involves a massive muscle defect in an unpredictable environment, subject to inflammatory waves weeks to months after irradiation, which delay healing and predispose the patient to the development of fibrous scar tissue. In this study, we investigated the long-term effect of local injections of bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery, to treat muscle necrosis in a large animal model. METHODS: Three months after irradiation to the rump, minipigs were treated by excision of necrotic muscle tissue, vascularized flap surgery, and four injections with or without local autologous BM-MSCs, performed weekly. The quality of the muscle wound healing was examined 1 year post-surgery. RESULTS: The skeletal muscle surgery without MSC treatment led to permanent deposition of collagen 1 and 3, decreased myofiber diameter, failed muscle fiber regeneration, a reduced number of capillaries, and the accumulation of high calcium and fat. In animals treated by surgery and MSC injections, these indicators were substantially better and demonstrated established regeneration. MSC therapy acts at several levels by stimulating growth factors such as VEGF, which is involved in angiogenesis and satellite cell pool maintenance, and creating a macrophage M1/M2 balance. CONCLUSION: Thus, cell therapy using BM-MSCs is an effective and safe way to improve recovery of irradiation-induced skeletal muscle damage without signs of long-term degeneration.

Autologous Bone Marrow Mesenchymal Stem Cells Improve the Quality and Stability of Vascularized Flap Surgery of Irradiated Skin in Pigs.

Cutaneous radiation syndrome has severe long-term health consequences. Because it causes an unpredictable course of inflammatory waves, conventional surgical treatment is ineffective and often leads to a fibronecrotic process. Data about the long-term stability of healed wounds, with neither inflammation nor resumption of fibrosis, are lacking. In this study, we investigated the effect of injections of local autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery for skin necrosis, in a large-animal model. Three months after irradiation overexposure to the rump, minipigs were divided into three groups: one group treated by simple excision of the necrotic tissue, the second by vascularized-flap surgery, and the third by vascularized-flap surgery and local autologous BM-MSC injections. Three additional injections of the BM-MSCs were performed weekly for 3 weeks. The quality of cutaneous wound healing was examined 1 year post-treatment. The necrotic tissue excision induced a pathologic scar characterized by myofibroblasts, excessive collagen-1 deposits, and inadequate vascular density. The vascularized-flap surgery alone was accompanied by inadequate production of extracellular matrix (ECM) proteins (decorin, fibronectin); the low col1/col3 ratio, associated with persistent inflammatory nodules, and the loss of vascularization both attested to continued immaturity of the ECM. BM-MSC therapy combined with vascularized-flap surgery provided mature wound healing characterized by a col1/col3 ratio and decorin and fibronectin expression that were all similar to that of nonirradiated skin, with no inflammation, and vascular stability. In this preclinical model, vascularized flap surgery successfully and lastingly remodeled irradiated skin only when combined with BM-MSC therapy. Stem Cells Translational Medicine 2018:569-582.

Radiocurability by targeting tumor necrosis factor-alpha using a bispecific antibody in carcinoembryonic antigen transgenic mice.

PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) enhances radiotherapy (RT) killing of tumor cells in vitro and in vivo. To overcome systemic side effects, we used a bispecific antibody (BsAb) directed against carcinoembryonic antigen (CEA) and TNF-alpha to target this cytokine in a CEA-expressing colon carcinoma. We report the evaluation of this strategy in immunocompetent CEA-transgenic mice. METHODS AND MATERIALS: The murine CEA-transfected colon carcinoma MC-38 was used for all experiments. In vitro, clonogenic assays were performed after RT alone, TNF-alpha alone, and RT plus TNF-alpha. In vivo, the mice were randomly assigned to treatment groups: control, TNF-alpha, BsAb, BsAb plus TNF-alpha, RT, RT plus TNF-alpha, and RT plus BsAb plus TNF-alpha. Measurements of endogenous TNF-alpha mRNA levels and evaluation of necrosis (histologic evaluation) were assessed per treatment group. RESULTS: In vitro, combined RT plus TNF-alpha resulted in a significant decrease in the survival fraction at 2 Gy compared with RT alone (p < 0.00001). In vivo, we observed a complete response in 5 (50%) of 10, 2 (20%) of 10, 2 (18.2%) of 11, and 0 (0%) of 12 treated mice in the RT plus BsAb plus TNF-alpha, RT plus TNF-alpha, RT alone, and control groups, respectively. This difference was statistically significant when TNF-alpha was targeted with the BsAb (p = 0.03). The addition of exogenous TNF-alpha to RT significantly increased the endogenous TNF-alpha mRNA level, particularly when TNF-alpha was targeted with BsAb (p < 0.01). The percentages of necrotic area were significantly augmented in the RT plus BsAb plus TNF-alpha group. CONCLUSION: These results suggest that targeting TNF-alpha with the BsAb provokes RT curability in a CEA-expressing digestive tumor syngenic model and could be considered as a solid rationale for clinical trials.

Collapse of skin antioxidant status during the subacute period of cutaneous radiation syndrome: a case report.

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.

Caffeic acid phenethyl ester modifies the Th1/Th2 balance in ileal mucosa after gamma-irradiation in the rat by modulating the cytokine pattern.

AIM: To pharmacologically modulate Th polarization in the ileum exposed to ionizing radiation by using the immuno-modulatory/apoptotic properties of Caffeic Acid Phenethyl Ester (CAPE). METHODS: Rats received CAPE (30 mg/kg) treatment ip 15 min prior to intestinal 10 Gy gamma-irradiation and once a day for a 6 d period after irradiation. Expression of genes implicated in Th differentiation in ileal mucosa (IL-23/IL-12Rbeta2), Th cytokine responses (IFN-gamma, IL-2, IL-4, IL-13), Th migratory behaviour (CXCR3, CCR5, CCR4), Th signalling suppressors (SOCS1, SOCS3), transcription factor (T-Bet, GATA-3) and apoptosis (FasL/Fas, TNF/TNFR, XIAP, Bax, caspase-3) was analyzed by RT-PCR 6 h and 7 d post-irradiation. CD4(+) and TUNEL positive cells were visualized by immunostaining. RESULTS: The expression of Th1-related cytokine/chemokine receptors (IFN-gamma, IL-2, CXCR3, CCR5) was repressed at 7 d post-irradiation while Th2 cell cytokine/chemokines (IL-4, IL-13, CCR4) were not repressed or even upregulated. The irradiation-induced Th2 profile was confirmed by the upregulation of both Th2-specific transcription factor GATA-3 and SOCS3. Although an apoptosis event occurred 6 h after 10 Gy of intestinal gamma-irradiation, apoptotic mediator analysis showed a tendency to apoptotic resistance 7 d post-irradiation. CAPE amplified apoptotic events at 6 h and normalized Bax/FasL expressions at 7 d. CONCLUSION: CAPE prevented the ileal Th2 immune response by modulating the irradiation-influenced cytokine environment and apoptosis.

Flagellin preconditioning enhances the efficacy of mesenchymal stem cells in an irradiation-induced proctitis model.

The success of mesenchymal stem cell transplantation for proctitis depends not only on cell donors but also on host microenvironmental factors, which play a major role in conditioning mesenchymal stem cell immunosuppressive action and repair. This study sought to determine if flagellin, a TLR5 ligand, can enhance the mesenchymal stem cell treatment efficacy in radiation-induced proctitis. With the use of a colorectal model of 27 Gy irradiation in rats, we investigated and compared the effects on immune capacity and remodeling at 28 d after irradiation of the following: 1) systemic mesenchymal stem cell (5 × 10(6)) administration at d 7 after irradiation, 2) administration of flagellin at d 3 and systemic mesenchymal stem cell administration at d 7, and 3) in vitro preconditioning of mesenchymal stem cells with flagellin, 24 h before their administration on d 7. The mucosal CD8(+) T cell population was normalized after treatment with flagellin-preconditioned mesenchymal stem cells or flagellin plus mesenchymal stem cells, whereas mesenchymal stem cells alone did not alter the radiation-induced elevation of CD8(+) T cell frequency. Mesenchymal stem cell treatment returned the irradiation-elevated frequency of CD25(+) cells in the mucosa-to-control levels, whereas both flagellin-preconditioned mesenchymal stem cell and flagellin-plus-mesenchymal stem cell treatment each significantly increased not only CD25(+) cell frequency but also forkhead box p3 and IL-2Rα expression. Specifically, IL-10 was overexpressed after flagellin-preconditioned mesenchymal stem cell treatment. Analysis of collagen expression showed that the collagen type 1/collagen type 3 ratio, an indicator of wound-healing maturation, was low in the irradiated and mesenchymal stem cell-treated groups and returned to the normal level only after the flagellin-preconditioned mesenchymal stem cell treatment. This was associated with a reduction in myofibroblast accumulation. In a proctitis model, flagellin-preconditioned mesenchymal stem cells improved colonic immune capacity and enhanced tissue remodeling.

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.

Expression of matrix metalloproteinases and tissue inhibitor metalloproteinases increases in X-irradiated rat ileum despite the disappearance of CD8a T cells.

AIM: To investigate their expression and activity in the rat ileum after exposure to ionizing radiation along with that of the cellular effectors and molecular mediators involved in the regulation of MMPs. METHODS: Rats were exposed to a single 10-Gy dose of X-rays delivered to the abdomen. A combination of methods, such as zymography, immunohistochemistry and real time reverse transcriptase-polymerase chain reaction, were used to localize and quantify MMPs and the molecules involved in MMP activating and inhibitory pathways (plasmin/plasminogen, TIMPs), CD8+, as well as inflammatory (interleukin (IL)-1beta, IL-8, tumor necrosis factor-alpha, TNF-alpha) and fibrogenic mediators (transforming growth factor-beta1-3) within ileal tissue at 1, 3, and 7 d after irradiation. RESULTS: A marked increase in MMP-2 and -14 mRNA and protein levels associated with an increased activity of MMP-2 was observed in irradiated ileal tissue. MMP-2 and -14 expression was mainly observed in inflammatory, epithelial, and mesenchymal cells, whereas a slight increase in MMP-3 expression was detected in the few infiltrating macrophages at d 1 after irradiation. Conversely, MMP-1, -7, and -9 mRNA levels were not found to be affected by abdominal irradiation. Irradiation was found to induce disappearance of CD8+ cells. Furthermore, we have observed that TNF-alpha and IL-1beta protein levels increased 6 h after irradiation, whereas those of IL-8 only increased after 3 d and was concomitant with neutrophil infiltration. In addition, the expressions of molecules involved in MMP activating and inhibitory pathways (urokinase-type plasminogen activator and tissue-type plasminogen activator; TIMP-1, TIMP-2, and plasminogen activator-inhibitor-1) were found to be increased after abdominal irradiation. CONCLUSION: This study showed that abdominal irradiation induces an acute remodeling of the ileum associated with an increased expression of MMPs and TIMPs that do not involve CD8+ T cells but involve mesenchymal and epithelial cells, although to a lesser extent, and probably even soluble inflammatory and fibrogenic mediators.

Therapeutic potential of gingival fibroblasts for cutaneous radiation syndrome: comparison to bone marrow-mesenchymal stem cell grafts.

Mesenchymal stem cell (MSC) therapy has recently been investigated as a potential treatment for cutaneous radiation burns. We tested the hypothesis that injection of local gingival fibroblasts (GFs) would promote healing of radiation burn lesions and compared results with those for MSC transplantation. Human clinical- grade GFs or bone marrow-derived MSCs were intradermally injected into mice 21 days after local leg irradiation. Immunostaining and real-time PCR analysis were used to assess the effects of each treatment on extracellular matrix remodeling and inflammation in skin on days 28 and 50 postirradiation. GFs induced the early development of thick, fully regenerated epidermis, skin appendages, and hair follicles, earlier than MSCs did. The acceleration of wound healing by GFs involved rearrangement of the deposited collagen, modification of the Col/MMP/TIMP balance, and modulation of the expression and localization of tenascin-C and of the expression of growth factors (VEGF, EGF, and FGF7). As MSC treatment did, GF injection decreased the irradiation-induced inflammatory response and switched the differentiation of macrophages toward an M2-like phenotype, characterized by CD163(+) macrophage infiltration and strong expression of arginase-1. These findings indicate that GFs are an attractive target for regenerative medicine, for easier to collect, can grow in culture, and promote cutaneous wound healing in irradiation burn lesions.

Acute induction of inflammatory cytokine expression after gamma-irradiation in the rat: effect of an NF-kappaB inhibitor.

PURPOSE: The pathologic changes within the intestinal muscle layer may be at the origin of the cytokines that account for acute radiation-induced inflammation. We were specifically interested in evaluating the efficacy of an inhibitor of nuclear transcription factor kappa B (NF-kappaB) activation that is involved in regulating cytokine expression. METHODS AND MATERIALS: Cytokine expression was analyzed in the ileal muscularis layer by reverse transcriptase-polymerase chain reaction (RT-PCR) at 3 h, 6 h, and 3 days after a 10-Gy gamma whole-body irradiation of rats. Caffeic acid phenethyl ester (CAPE) was injected intraperitoneally (30 mg/kg) 15 min before irradiation and once a day for 3 days. RESULTS: Interleukin (IL)-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNA increased at 3 h and 6 h after irradiation, and expression of IL-6 and IL-8 was elevated at 3 days. On the other hand, levels of the anti-inflammatory cytokine IL-10 were markedly lower on Day 3. Overexpression of IL-6 on Day 3 was combined with upregulation of the IL-6 receptors (gp130/gp80) and suppressor of cytokine signaling-3 (SOCS3) genes. CAPE treatment did not significantly change IL-1beta or TNF-alpha expressions in the irradiated rats; it increased IL-10 expression at 6 h but had no effect on it on Day 3. CAPE treatment inhibited the radiation-induced expression of IL-6, IL-6 receptors (IL-6rs), and SOCS3 at 3 days. CONCLUSION: In vivo, irradiation induced a cascade of inflammatory responses that involved the transcription factor NF-kappaB; this inflammation was reduced by CAPE treatment.

Gene expression profile in human late radiation enteritis obtained by high-density cDNA array hybridization.

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.

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 colon's 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.