Novel chimeric TLR2/NOD2 agonist CL429 exhibited significant radioprotective effects in mice

Severe ionizing radiation causes the acute lethal damage of haematopoietic system and gastrointestinal tract. Here, we found CL429, the novel chimeric TLR2/NOD2 agonist, exhibited significant radioprotective effects in mice. CL429 increased mice survival, protected mice against the lethal damage of haematopoietic system and gastrointestinal tract. CL429 was more effective than equivalent amounts of monospecific (TLR2 or NOD2) and combination (TLR2 + NOD2) of molecules in preventing radiation-induced death. The radioprotection of CL429 was mainly mediated by activating TLR2 and partially activating NOD2. CL429-induced radioprotection was largely dependent on the activation of TLR2-MyD88-NF-κB signalling pathway. In conclusion, the data suggested that the co-activation of TLR2 and NOD2 could induce significant synergistic radioprotective effects and CL429 might be a potential high-efficiency selective agent.     

Macrophage-derived exosomes mediate silica-induced pulmonary fibrosis by activating fibroblast in an endoplasmic reticulum stress-dependent manner

Macrophages play a key role in silicosis, and exosomes are potent mediators of intercellular communication. This suggests that macrophage-derived exosomes have a potential contribution to the pathogenesis of silicosis. To investigate whether macrophage-derived exosomes promote or inhibit lung fibrosis, in vitro, silica-exposed macrophage-derived exosomes (SiO₂-Exos) were collected and cocultured with fibroblasts. The expression of collagen I and α-SMA was evaluated. Furthermore, the endoplasmic reticulum (ER) stress markers BIP, XBP1s and P-eIF2α were assessed after treatment with or without the ER stress inhibitor 4-PBA. In vivo, mice were pre-treated with the exosome secretion inhibitor GW4869 prior to silica exposure. After sacrifice, lung tissues were histologically examined, and the expression of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in bronchoalveolar lavage fluid (BALF) was measured. The results showed that the expression of collagen I and α-SMA was up-regulated after treatment with SiO₂-Exos, accompanied by increased expression of BIP, XBP1s and P-eIF2α. Pre-treatment with 4-PBA reversed this effect. More importantly, an in vivo study demonstrated that pre-treatment with GW4869 decreased lung fibrosis and the expression of TNF-α, IL-1β and IL-6 in BALF. These results suggested that SiO₂-Exos are profibrogenic and that the facilitating effect is dependent on ER stress.     

Monophosphoryl Lipid A‐Adjuvanted Virosomes with Ni‐Chelating Lipids for Attachment of Conserved Viral Proteins as Cross‐Protective Influenza Vaccine

Induction of CD8⁺ cytotoxic T cells (CTLs) to conserved internal influenza antigens, such as nucleoprotein (NP), is a promising strategy for the development of cross‐protective influenza vaccines. However, influenza NP protein alone cannot induce CTL immunity due to its low capacity to activate antigen‐presenting cells (APCs) and get access to the MHC class I antigen processing pathway. To facilitate the generation of NP‐specific CTL immunity the authors develop a novel influenza vaccine consisting of virosomes with the Toll‐like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) and the metal‐ion‐chelating lipid DOGS‐NTA‐Ni incorporated in the membrane. In vitro, virosomes with incorporated MPLA induce stronger activation of APCs than unadjuvanted virosomes. Virosomes modified with DOGS‐NTA‐Ni show high conjugation efficacy for his‐tagged proteins and facilitate efficient uptake of conjugated proteins by APCs. Immunization of mice with MPLA‐adjuvanted virosomes with attached NP results in priming of NP‐specific CTLs while MPLA‐adjuvanted virosomes with admixed NP are inefficient in priming CTLs. Both vaccines induce equally high titers of NP‐specific antibodies. When challenged with heterosubtypic influenza virus, mice immunized with virosomes with attached or admixed NP are protected from severe weight loss. Yet, unexpectedly, they show more weight loss and more severe disease symptoms than mice immunized with MPLA‐virosomes without NP. Taken together, these results indicate that virosomes with conjugated antigen and adjuvant incorporated in the membrane are effective in priming of CTLs and eliciting antigen‐specific antibody responses in vivo. However, for protection from influenza infection NP‐specific immunity appears not to be advantageous.     

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.     

Biological consequence of nuclear versus cytoplasmic decays of 125I: cysteamine as a radioprotector against Auger cascades in vivo

When the radionuclide 125I is localized in mouse testis as 125I-iododeoxyuridine (an analogue of thymidine) and incorporated into the DNA of spermatogonial cells, the cytocidal effects are as severe as those due to densely ionizing alpha particles. In contrast, 125I confined to the cytoplasm of these cells is much less radiotoxic, the efficacy being the same as for selective irradiation of the testis with sparsely ionizing external X rays. The biological effects, in both cases, are strongly mitigated upon pretreatment of the testes with very small amounts (0.75 microgram) of cysteamine, a radioprotector. These findings suggest an important role for such chemical agents in radiation protection and in understanding the mechanisms of radiation damage involving radionuclides incorporated in tissue.     

Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury

Activation of macrophages via toll-like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non-pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFkappaB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2-/-) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild-type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin-1beta mRNA. In TLR2-/- mice, locomotor recovery also was impaired relative to SCI wild-type controls and novel patterns of myelin pathology existed within ventromedial white matter--an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post-injury sequelae and perhaps in regulating inflammation and gliosis after SCI.     

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.     

Cutting edge: high-mobility group box 1 preconditioning protects against liver ischemia-reperfusion injury

High mobility group box 1 (HMGB1) is a NF released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. Here we demonstrate that in contrast to the proinflammatory role of HMGB1, preconditioning with HMGB1 results in protection following hepatic ischemia/reperfusion (I/R). Pretreatment of mice with HMGB1 significantly decreased liver damage after I/R. The protection observed in mice pretreated with HMGB1 was associated with a higher expression of IL-1R-associated kinase-M, a negative regulator of TLR4 signaling, compared with controls. We thus explored the possibility that HMGB1 preconditioning was mediated through TLR4 activation. HMGB1 preconditioning failed to provide protection in TLR4 mutant (C3H/HeJ) mice, but successfully reduced damage in TLR4 wild-type (C3H/HeOuj) mice. Our studies demonstrate that in contrast to the role of HMGB1 as an early mediator of inflammation and organ damage in hepatic I/R, HMGB1 preconditioning can be protective.     

Protective effects of activated vitamin D receptor on radiation-induced intestinal injury

Radiation-induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) -induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti-injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation-induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation-induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation-induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.     

Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism

The intestinal epithelium is sensitive to radiation injury. Damage to the intestinal epithelium is dose limiting in radiation therapy of abdominal cancers. There is a need for agents that can be given before radiation therapy to protect the intestinal epithelium. C57BL6 mice were subjected to 12 Gy of total body radiation. Some mice received intraperitoneal hyaluronic acid (HA) before radiation. Mice were killed 6 h after radiation to assess radiation-induced apoptosis in the intestine; other mice were killed at 84 h to assess crypt survival. Total body radiation (12 Gy) resulted in increased expression of HA synthases and HA in the intestine and increased plasma HA (5-fold). Intraperitoneal injection of HA (30 mg/kg) before radiation resulted in a 1.8-fold increase in intestinal crypt survival and a decrease in radiation-induced apoptosis. The radioprotective effects of HA were not seen in Toll-like receptor 4 (TLR4)- or cyclooxygenase-2 (COX-2)-deficient mice. Intraperitoneal injection of HA induced a 1.5-fold increase in intestinal COX-2 expression, a 1.5-fold increase in intestinal PGE?, and the migration of COX-2-expressing mesenchymal stem cells from the lamina propria in the villi to the lamina propria near the crypt. We conclude that 1) radiation induces increased HA expression through inducing HA synthases, 2) intraperitoneal HA given before radiation reduces radiation-induced apoptosis and increases crypt survival, and 3) these radioprotective effects are mediated through TLR4, COX-2, and the migration of COX-2-expressing mesenchymal stem cells.     

Lasting effects of an impairment of Th1-like immune response in γ-irradiated mice: A resemblance between irradiated mice and aged mice

Although one of the several chronic effects of ionizing radiation is aging, there are no experimental data on radiation-induced immunological aging. The most interesting change in aging was a helper T (Th) 1/Th2 imbalance. We investigated chronic effect on immune responses after ionizing radiation and its effects in irradiated mice were compared with those of aged mice. The 2-month-old mice received a whole-body irradiation of 5 Gy. At 6 months after irradiation, we compared the immune functions of the irradiated mice with those of normal mice of the same age and with those of older. Interferon (IFN)-γ and antigen-specific immunoglobulin (Ig)G2a level were lower in the irradiated mice than in normal mice of same age, showing similar levels to those of old normal mice. In contrast, interleukin (IL)-4 and IL-5 and antigen-specific IgG1 level were increased in irradiated mice when compared with the same aged-normal mice. Next, we investigated the low expression of IL-12p70, IL-12 receptors and IL-18 receptors in irradiated and old mice. Also, the decrease of natural killer cell activity was intensified in the irradiated mice, showing lower than values to those of old mice. Interestingly, in irradiated mice, the absolute numbers and the percentages of natural killer (NK) cells was extremely decreased. But the absolute numbers of Th cells and cytotoxic T (Tc) cells in old mice were significantly decreased. In conclusion, an immunological imbalance by the whole-body irradiation of 5 Gy induces to persist in the long term, resulting in the similar results with aging. Our results suggest that the downregulation of the Th1-like immune response shown in old mice rapidly occurred through exposure of ionizing radiation.     

Absence of TLR4 Reduces Neurovascular Unit and Secondary Inflammatory Process after Traumatic Brain Injury in Mice

Background

Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. Toll-like receptors (TLRs) are signaling receptors in the innate immune system, although emerging evidence indicates their role in brain injury. We have therefore investigated the role played by TLR4 signaling pathway in the development of mechanisms of secondary inflammatory process in traumatic brain injury (TBI) differ in mice that lack a functional TLR4 signaling pathway.

Methods/Principal Findings

Controlled cortical impact injury was performed on TLR4 knockout (KO) mice (C57BL/10ScNJ) and wild-type (WT) mice (C57BL/10ScNJ). TBI outcome was evaluated by determination of infarct volume and assessment of neurological scores. Brains were collected at 24 h after TBI. When compared to WT mice, TLR4 KO mice had lower infarct volumes and better outcomes in neurological and behavioral tests (evaluated by EBST and rotarod test). Mice that lacked TLR4 had minor expression of TBI-induced GFAP, Chymase, Tryptase, IL-1β, iNOS, PARP and Nitrotyrosine mediators implicated in brain damage. The translocation of expression of p-JNK, IκB-α and NF-κB pathway were also lower in brains from TLR4 KO mice. When compared to WT mice, resulted in significant augmentation of all the above described parameters. In addition, apoptosis levels in TLR4 KO mice had minor expression of Bax while on the contrary with Bcl-2.

Conclusions/Significance

Our results clearly demonstrated that absence of TLR4 reduces the development of neuroinflammation, tissues injury events associated with brain trauma and may play a neuroprotective role in TBI in mice.     

Testosterone reduces macrophage expression in the mouse of toll-like receptor 4, a trigger for inflammation and innate immunity

Though gender-based differences in the development of protective or pathological adaptive host responses have been widely noted, it is becoming apparent that sex may also influence the early perception of microbial challenges and the generation of inflammatory immune responses. These differences may be due to the actions of reproductive hormones, and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions, including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study, we have investigated the acute effects of testosterone on the level of expression of a key trigger for inflammation and innate immunity, Toll-like receptor 4 (TLR4), on isolated mouse macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in TLR4 expression and sensitivity to a TLR4-specific ligand. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression and endotoxin susceptibility. We report that orchidectomized mice were significantly more susceptible to endotoxic shock and show that macrophages isolated from these animals have significantly higher TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, these effects were not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data may represent an important mechanism underlying the immunosuppressive effects of testosterone.     

Ionizing radiation stimulates secretion of pro-inflammatory cytokines: dose–response relationship,mechanisms and implications

In previous studies we showed a marked increase in secretion of inflammatory cytokines TNFα and interleukin (IL)-1β by mouse macrophages in response to different doses of ionizing radiation (IR). Here we show the stimulation of IL-12 and IL-18 secretion by mouse peritoneal macrophages after whole-body irradiation with exploration of the possible mechanisms and implications in cancer radiotherapy. Both low (0.075 Gy) and high (2 Gy) doses of IR were found to cause sustained stimulation of IL-12 and IL-18 secretion by mouse macrophages; this paralleled the activation of NF-κB as well as up-regulated expression of CD14 and TLR4–MD2 on the macrophage surface and MyD88 in the cytoplasm. The expression of CD14, TLR4–MD2 and MyD88 increased in a dose-dependent manner from radiation doses between 0.05 and 2 Gy. The secretion of IL-12 and IL-18 showed a dose-dependent increase from doses between 0.05 and 4 Gy. It is concluded that IR can stimulate the secretion of IL-12 and IL-18 presumably via activation of the Toll signaling pathway in macrophages. The potential harmful effect of repeated doses of radiation used in radiotherapy for certain cancers is discussed. Yu-Xing Shan and Shun-Zi Jin contributed equally to the present work.     

The Ultra-Potent and Selective TLR8 Agonist VTX-294 Activates Human Newborn and Adult Leukocytes

Background

Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed.

Methods

TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry).

Results

VTX-294 was ∼100x more active on TLR8- than TLR7-transfected HEK cells (EC₅₀, ∼50 nM vs. ∼5700 nM). VTX-294-induced TNF and IL-1β production were comparable in newborn cord and adult peripheral blood, while VTX-294 was ∼ 1 log more potent in inducing TNF and IL-1β production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1β responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs.

Conclusions

VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.     

Loss of Toll-Like Receptor 4 Function Partially Protects against Peripheral and Cardiac Glucose Metabolic Derangements During a Long-Term High-Fat Diet

Diabetes is a chronic inflammatory disease that carries a high risk of cardiovascular disease. However, the pathophysiological link between these disorders is not well known. We hypothesize that TLR4 signaling mediates high fat diet (HFD)-induced peripheral and cardiac glucose metabolic derangements. Mice with a loss-of-function mutation in TLR4 (C3H/HeJ) and age-matched control (C57BL/6) mice were fed either a high-fat diet or normal diet for 16 weeks. Glucose tolerance and plasma insulin were measured. Protein expression of glucose transporters (GLUT), AKT (phosphorylated and total), and proinflammatory cytokines (IL-6, TNF-α and SOCS-3) were quantified in the heart using Western Blotting. Both groups fed a long-term HFD had increased body weight, blood glucose and insulin levels, as well as impaired glucose tolerance compared to mice fed a normal diet. TLR4-mutant mice were partially protected against long-term HFD-induced insulin resistance. In control mice, feeding a HFD decreased cardiac crude membrane GLUT4 protein content, which was partially rescued in TLR4-mutant mice. TLR4-mutant mice fed a HFD also had increased expression of GLUT8, a novel isoform, compared to mice fed a normal diet. GLUT8 content was positively correlated with SOCS-3 and IL-6 expression in the heart. No significant differences in cytokine expression were observed between groups, suggesting a lack of inflammation in the heart following a HFD. Loss of TLR4 function partially restored a healthy metabolic phenotype, suggesting that TLR4 signaling is a key mechanism in HFD-induced peripheral and cardiac insulin resistance. Our data further suggest that TLR4 exerts its detrimental metabolic effects in the myocardium through a cytokine-independent pathway.