Imaging Pulmonary NF-kappaB Activation and Therapeutic Effects of MLN120B and TDZD-8

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions

Genome-Wide Identification of Susceptibility Alleles for Viral Infections through a Population Genetics Approach

Viruses have exerted a constant and potent selective pressure on human genes throughout evolution. We utilized the marks left by selection on allele frequency to identify viral infection-associated allelic variants. Virus diversity (the number of different viruses in a geographic region) was used to measure virus-driven selective pressure. Results showed an excess of variants correlated with virus diversity in genes involved in immune response and in the biosynthesis of glycan structures functioning as viral receptors; a significantly higher than expected number of variants was also seen in genes encoding proteins that directly interact with viral components. Genome-wide analyses identified 441 variants significantly associated with virus-diversity; these are more frequently located within gene regions than expected, and they map to 139 human genes. Analysis of functional relationships among genes subjected to virus-driven selective pressure identified a complex network enriched in viral products-interacting proteins. The novel approach to the study of infectious disease epidemiology presented herein may represent an alternative to classic genome-wide association studies and provides a large set of candidate susceptibility variants for viral infections

IKKalpha Contributes to Canonical NF-kappaB Activation Downstream of Nod1-Mediated Peptidoglycan Recognition

BACKGROUND: During pathogen infection, innate immunity is initiated via the recognition of microbial products by pattern recognition receptors and the subsequent activation of transcription factors that upregulate proinflammatory genes. By controlling the expression of cytokines, chemokines, anti-bacterial peptides and adhesion molecules, the transcription factor nuclear factor-kappa B (NF-kappaB) has a central function in this process. In a typical model of NF-kappaB activation, the recognition of pathogen associated molecules triggers the canonical NF-kappaB pathway that depends on the phosphorylation of Inhibitor of NF-kappaB (IkappaB) by the catalytic subunit IkappaB kinase beta (IKKbeta), its degradation and the nuclear translocation of NF-kappaB dimers. METHODOLOGY: Here, we performed an RNA interference (RNAi) screen on Shigella flexneri-induced NF-kappaB activation to identify new factors involved in the regulation of NF-kappaB following infection of epithelial cells by invasive bacteria. By targeting a subset of the human signaling proteome, we found that the catalytic subunit IKKalpha is also required for complete NF-kappaB activation during infection. Depletion of IKKalpha by RNAi strongly reduces the nuclear translocation of NF-kappaB p65 during S. flexneri infection as well as the expression of the proinflammatory chemokine interleukin-8. Similar to IKKbeta, IKKalpha contributes to the phosphorylation of IkappaBalpha on serines 32 and 36, and to its degradation. Experiments performed with the synthetic Nod1 ligand L-Ala-D-gamma-Glu-meso-diaminopimelic acid confirmed that IKKalpha is involved in NF-kappaB activation triggered downstream of Nod1-mediated peptidoglycan recognition. CONCLUSIONS: Taken together, these results demonstrate the unexpected role of IKKalpha in the canonical NF-kappaB pathway triggered by peptidoglycan recognition during bacterial infection. In addition, they suggest that IKKalpha may be an important drug target for the development of treatments that aim at limiting inflammation in bacterial infection