The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity.

Using DNA microarrays together with quantitative proteomic techniques (ICAT reagents, two-dimensional DIGE, and MS), we evaluated the correlation of mRNA and protein levels in two hematopoietic cell lines representing distinct stages of myeloid differentiation, as well as in the livers of mice treated for different periods of time with three different peroxisome proliferative activated receptor agonists. We observe that the differential expression of mRNA (up or down) can capture at most 40% of the variation of protein expression. Although the overall pattern of protein expression is similar to that of mRNA expression, the incongruent expression between mRNAs and proteins emphasize the importance of posttranscriptional regulatory mechanisms in cellular development or perturbation that can be unveiled only through integrated analyses of both proteins and mRNAs. Molecular & Cellular Proteomics 3:960 -969, 2004.Genome-wide mRNA expression profiling by means of DNA microarrays has proven to be a powerful approach in characterizing the changes in biological processes such as disease states, developmental stages, and responses to drugs or genetic perturbations (1). However, DNA arrays measure only the changes at the mRNA level. Most biological functions are executed by the proteins rather than mRNAs. While the expression of many genes is controlled at the transcriptional level, other genes also employ posttranscriptional regulation processes involving mRNA stability, translation initiation, and protein stability. An important issue is the extent to which the changing expression patterns of mRNAs reflect corresponding changes in their cognate proteins. Recent advances in quantitative proteomics, especially the application of ICAT reagents in conjunction with MS, have made possible simultaneous quantitative comparison of hundreds of proteins between two complex mixtures (2). Integrated analyses of mRNA and protein expression data by concurrent measurement of both have revealed moderate to poor correlation in yeast and Halobacteria (3-5). Discordant expression of protein and mRNA was also observed in lung adenocarcinomas (6). However, these analyses examined only one aspect of a biological system, i.e. the steady-state levels of mRNAs and proteins. Another important aspect that concerns the kinetic process of perturbation and how the correlation of mRNA and protein evolves during this process was not addressed. Here, we evaluated the correlation of mRNA and protein expression in mammalian systems under two experimental conditions. In the first, we compared steady-state levels of mRNAs and proteins between two related cell lines representing distinct hematopoietic stages, i.e. multipotent myeloid precursors versus lineage-committed promyelocytic cells. In the second condition, we used a mouse model to demonstrate the kinetic changes in liver mRNA and protein levels in response to treatment with three different drugs. In both cases, we observed a moderate correlation between mRNA and protein levels with the expression of m...

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.