The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and p53 Networks

In recent years the functions that the p53 tumor suppressor plays in human biology have been greatly extended beyond “guardian of the genome.” Our studies of promoter response element sequences targeted by the p53 master regulatory transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human Toll-like receptor (TLR) gene expression. The TLR gene family mediates innate immunity to a wide variety of pathogenic threats through recognition of conserved pathogen-associated molecular motifs. Using primary human immune cells, we have examined expression of the entire TLR gene family following exposure to anti-cancer agents that induce the p53 network. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors. However, there is considerable inter-individual variability. Most of the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites. Importantly, the integration of the TLR gene family into the p53 network is unique to primates, a recurrent theme raised for other gene families in our previous studies. Furthermore, a polymorphism in a TLR8 response element provides the first human example of a p53 target sequence specifically responsible for endogenous gene induction. These findings—demonstrating that the human innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress—have many implications for health and disease, as well as for understanding the evolution of damage and p53 responsive networks

TLR9 Agonist Protects Mice from Radiation-Induced Gastrointestinal Syndrome

Radiation-induced gastrointestinal syndrome (RIGS) is due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of mucosal barrier, bacterial invasion, inflammation and sepsis. Intestinal macrophages could recognize invading bacterial DNA via TLR9 receptors and transmit regenerative signals to the neighboring crypt. We therefore investigated whether systemic administration of designer TLR9 agonist could ameliorate RIGS by activating TLR9.Male C57Bl6 mice were distributed in four experimental cohorts, whole body irradiation (WBI) (8.4-10.4 Gy), TLR9 agonist (1 mg/kg s.c.), 1 h pre- or post-WBI and TLR9 agonist+WBI+iMyd88 (pretreatment with inhibitory peptide against Myd88). Animals were observed for survival and intestine was harvested for histological analysis. BALB/c mice with CT26 colon tumors in abdominal wall were irradiated with 14 Gy single dose of whole abdominal irradiation (AIR) for tumor growth study.Mice receiving pre-WBI TLR9 agonist demonstrated improvement of survival after 10.4 Gy (p<0.03), 9.4 Gy (p<0.008) and 8.4 Gy (p<0.002) of WBI, compared to untreated or iMyd88-treated controls. Post-WBI TLR9 agonist mitigates up to 8.4 Gy WBI (p<0.01). Histological analysis and xylose absorption test demonstrated significant structural and functional restitution of the intestine in WBI+TLR9 agonist cohorts. Although, AIR reduced tumor growth, all animals died within 12 days from RIGS. TLR9 agonist improved the survival of mice beyond 28 days post-AIR (p<0.008) with significant reduction of tumor growth (p<0.0001).TLR9 agonist treatment could serve both as a prophylactic or mitigating agent against acute radiation syndrome and also as an adjuvant therapy to increase the therapeutic ratio of abdominal Radiation Therapy for Gastro Intestinal malignancies

Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs).Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-kappaB activation, but were potent in eliciting EGFR signaling in a TGF-alpha-related pathway in HCECs. Concomitant with the lack of NF-kappaB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling.Our data suggest that inflammatory response mediated by NF-kappaB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues

HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin

Selective protection of normal tissues from radiation damage by activation of Toll-like receptor 5 signaling

The toxicity of ionizing radiation (IR) is associated with the development of acute radiation syndrome primarily involving damage to the highly radiosensitive gastrointestinal (GI) tract and the hematopoietic (HP) system (1). Development of radioprotectants has been historically focused on agents that either reduce the degree of a direct damage (e.g., anti-oxidants) or stimulate tissue recovery and regeneration (e.g., cytokines) (2). These efforts have not produced a useful radiation countermeasure approved with an acceptable safety profile that would be effective against both major components of acute radiation syndrome leaving radioprotection an unmet medical need. We have recently introduced a new type of radioprotectants with a distinctly different mechanism of action which does satisfy the necessary requirements and can be considered for biodefense and medical application. Our strategy for radioprotection is based on the observation that massive cell loss occurring in radiosensitive tissues exposed to IR occurs predominantly through apoptosis, which is almost universally suppressed in tumors. Among the variety of mechanisms acquired by tumors to avoid apoptosis, there are two most universal ones - suppression of pro-apoptotic p53 pathway and constitutive activation of pro-survival NF-kB pathway. Our approach to selective radioprotection involves the development of pharmacological agents able to activate those mechanisms of apoptosis suppression that are utilized by tumor cells in normal cells. First, we started with the development of p53 inhibitors which offered effective protection to the HP system known to suffer cell loss predominantly through the induction of p53-dependent apoptosis (3, 4). However, the pro-survival role of p53 found in GI tract (5) prevented us from using p53 inhibitors for protection from this component of acute radiation syndrome. To protect the GI tract from acute radiation syndrome, we used agents known to activate NF-kB, the pathway that is constitutively active in the majority of tumors. Specifically, we focused on NF-kB-activating factors of human intestinal microflora known to be involved in supporting viability of intestinal tissue (6). The radioprtotectant we recently described, CBLB502, was derived from bacterial (Salmonella) flagellin, the only known ligand of Toll-like receptor 5 (TLR5) which is expressed in the epithelial and endothelial cells of the intestine and presumably plays a role in the protection of the GI tract from parasitic infections. Studies with flagellin and its less immunogenic, non-toxic derivative, CBLB502, have shown each able to provide significant radioprotection and mitigation of radiation injury to both the HP and GI systems and improve survival of mice and rhesus macaques after exposure to lethal IR (7). Importantly, CBLB502 was found effective in protecting mice from IR applied locally (head and neck), the regimen better imitating the use of radiation in oncology clinic. Treatment with CBLB502 significantly enhanced expression of a number of cellular defense factors encoded by NF-κB-responsive genes such as superoxide dismutase 2 (SOD2), a well-known scavenger of reactive oxygen species, that is induced in the lamina propria of irradiated mice and primates. Furthermore, CBLB502 injection led to the induction of high levels of G-CSF and other radioprotective cytokines with only minor increase in pro-inflammatory factors, such as TNFα or IL1. Thus, the mechanism by which CBLB502 prevents radiation toxicity is multi-fold and involves mobilization of endogenous defense mechanisms, combining traditional radioprotective approaches (induction of antioxidants and regeneration-supporting cytokines) with suppression of apoptosis in radiosensitive cells. CBLB502-mediated radioprotection was found to be highly selective for normal cells and did not change tumor cell sensitivity to IR regardless of whether they express functional TLR5 or not. This selectivity is presumably explained by constitutive activation of NF-kB observed in the majority of tumors (8), rendering this mechanism unsuitable for targeting by radioprotectans. The selectivity of CBLB502-mediated radioprotection for normal cells was tested in vitro by colony formation assays. Characterization of a large panel of tumor-derived and normal human and mouse cell variants grown in culture indicated that only normal cells are susceptible for radioprotection activity of CBLB502. The normal tissue selectivity of CBLB502-mediated radioprotection was further confirmed in vivo in several mouse tumor models of experimental radiotherapy, including syngenic (C57BL6 mice bearing B16 melanomas) and xenogenic tumors (athymic nude mice bearing human HCT116 colon tumors). The tumor-bearing mice were subjected to fractioned radiation treatment (3 or 4 Gy for three consecutive days, 100% lethal cumulative dose) with and without CBLB502 treatment. In all of the models tested, the antitumor effect of irradiation was accompanied by the death of all irradiated mice of the control group within 2-3 weeks after IR while treatment with CBLB502 prevented radiation-induced mortality or prolonged survival providing no protection to the tumors (Figure). These results support the development of CBLB502 as a drug able to prevent adverse effects of radiotherapy through its ability to protect both the GI tract and the HP system. The high selectivity of CBLB502 for protection of normal cells makes it uniquely qualified for radioprotection (prophylaxis) and radiomitigation regimens during cancer therapy. Figure. (see PDF file) Athymic female mice were inoculated with human colorectal cancer cells HCT116. When tumors reached approximately 5 mm in diameter, mice received CBLB502 injections followed by 3.3 Gy of total body irradiation 30 min later. This treatment was repeated 3 times daily. Two other groups of mice were treated with CBLB502 alone or irradiated. Control mice received PBS injections as a vehicle control. V=π/6 x length x width x width. References 1. J. F. Weiss, M. R. Landauer, Toxicology 189, 1 (Jul 15, 2003). 2. S. J. Hosseinimehr, Drug Discov Today 12, 794 (Oct, 2007). 3. P. G. Komarov et al., Science 285, 1733 (Sep 10, 1999). 4. E. Strom et al., Nat Chem Biol 2, 474 (Sep, 2006). 5. E. A. Komarova et al., Oncogene 23, 3265 (Apr 22, 2004). 6. S. Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, R. Medzhitov, Cell 118, 229 (Jul 23, 2004). 7. L. G. Burdelya et al., Science 320, 226 (Apr 11, 2008). 8. M. Karin, Nature 441, 431 (May 25, 2006)