Pharmacologic activation of the innate immune system to prevent respiratory viral infections.

Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-ß and subsequent high-level induction of IFN-ß-dependent proteins, such as myxovirus resistance 1 (Mx1) and 2',5'-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-ß system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and "first responders" in the early stages of viral pandemics or bioterror attacks.

Refractoriness of interferon-beta signaling through NOD1 pathway in mouse respiratory epithelial cells using the anticancer xanthone compound.

AIM: To explore the possibility that nucleotide oligomerization domain 1 (NOD1) pathway involved in refractoriness of interferon-ß signaling in mouse respiratory epithelial cells induced by the anticancer xanthone compound, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). METHODS: C10 mouse bronchial epithelial cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, 100 g/mL streptomycin. Pathogen-free female BALB/c mice were used to explore the mechanisms of refractoriness of interferon-signaling. Mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells were collected and cultured. The amount of interferon (IFN)-inducible protein-10 (IP10/CXCL10), macrophage chemotactic protein (MCP1/CCL2) and interleukin (IL)-6 secreted by cells activated by DMXAA was quantified using enzyme-linked immunosorbent assay kits according to the instructions of the manufacturers. Total RNA was isolated from cells or nasal epithelium with RNeasy Plus Mini Kit, and cDNA was synthesized. Gene expression was checked using Applied Biosystems StepOne Real-Time Polymerase Chain Reaction System. Transfection of small interfering RNA (siRNA) control, NOD1 duplexed RNA oligonucleotides, and high-mobility group box 1/2/3 (HMGB1/2/3) siRNA was performed using siRNA transfection reagent. RESULTS: DMXAA activates IFN-ß pathway with high level of IFN-ß dependent antiviral genes including 2', 5'-oligoadenylate synthetase 1 and myxovirus resistance 1 in mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells. Activation of IFN-ß by DMXAA involved in NOD1, but not HMGB1/2/3 signal pathway demonstrated by siRNA. NOD1 pathway plays an important role in refractoriness of IFN-ß signaling induced by DMXAA in mouse C10 respiratory epithelial cells and BALB/c mice nasal epithelia. These data indicate that DMXAA is not well adapted to the intrinsic properties of IFN-ß signaling. Approaches to restore sensitivity of IFN-ß signaling by find other xanthone compounds may function similarly, could enhance the efficacy of protection from influenza pneumonia and potentially in other respiratory viral infections. CONCLUSION: NOD1 pathway may play an important role in refractoriness of IFN-ß signaling in mouse respiratory epithelial cells induced by DMXAA.

Phosphatidylinositol-3-kinase-Akt kinase and p42/p44 mitogen-activated protein kinases mediate neurotrophic and excitoprotective actions of a secreted form of amyloid precursor protein.

The alpha-secretase-derived form of the amyloid precursor protein (sAPPalpha), which is released from neurons in an activity-dependent manner, has been shown to promote long-term survival of hippocampal and cortical neurons in culture and can protect those neurons against excitotoxic and ischemic injury in culture and in vivo. The signal transduction pathway(s) activated by sAPPalpha has not been established. We now report that sAPPalpha activates the phosphatidylinositol-3-kinase (PI(3)K)-Akt kinase signaling pathway in cultured hippocampal neurons. sAPPalpha also stimulates phosphorylation of p42 (ERK1) and p44 (ERK2) mitogen-activated protein (MAP) kinases by a PI(3)K-independent pathway. Treatment of neurons with sAPPalpha protects them against death induced by trophic factor deprivation and exposure to glutamate, and these survival-promoting effects of sAPPalpha are abolished or attenuated when either PI(3)K or p42/p44 MAP kinases are selectively blocked. Exposure of neurons to sAPPalpha resulted in a decrease in the level of IkappaBbeta and an increase in NF-kappaB DNA binding activity, both of which were blocked by wortmannin, suggesting that the transcription factor NF-kappaB may be a downstream target of the PI(3)K-Akt pathway that may play a role in the cell survival-promoting action of sAPPalpha. These findings suggest that the PI(3)K-Akt pathway and p42/p44 MAP kinases mediate responses of neurons to sAPPalpha in physiological and pathological settings, with implications for synaptic plasticity and the pathogenesis of Alzheimer's disease.

Tumor necrosis factor alpha increases neuronal vulnerability to excitotoxic necrosis by inducing expression of the AMPA-glutamate receptor subunit GluR1 via an acid sphingomyelinase- and NF-kappaB-dependent mechanism.

Acid sphingomyelinase (ASMase) and NF-kappaB participate in tumor necrosis factor alpha (TNFalpha) signal transduction. Mice in which the genes encoding ASMase or the p50 subunit of NF-kappaB are disrupted have been reported to be less vulnerable than wild-type mice to focal brain ischemia. We now demonstrate selective diminution in expression of GluR1, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptor (AMPA-GluR) protein subunit, in these two groups of knockout mice. To confirm that neuronal GluR1 expression is regulated by ASMase and NF-kappaB, and to learn whether this regulation has pathophysiological significance, we treated cultured human NT2-N neurons with TNFalpha. This induced GluR1 expression and increased susceptibility of the neurons to kainate necrosis. Both induction of GluR1 and heightened vulnerability to kainate were blocked by inhibiting ASMase or by antisense knockdown of NF-kappaB p50. We conclude that TNFalpha can sensitize neurons to excitotoxic necrosis by inducing expression of GluR1 via an ASMase- and NF-kappaB-dependent mechanism. TNFalpha levels are frequently elevated during ischemia and other CNS diseases in which excitotoxicity contributes to neuronal loss. Our results suggest that inhibiting TNFalpha signal transduction will diminish neuronal necrosis in these diseases.