Role of p52 (NF-κB2) in LPS tolerance in a human B cell line

Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-κB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS), When such cells are precultured with a low amount of LPS (50 - 250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 mu g/ml) then the cytokine expression is strongly reduced, i.e, the cells have become tolerant. Western blot analysis of proteins of the NF-kappa B/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells, Also, gelshift analysis with the -605 kappa B motif Of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells - a complex that is supershifted with an anti-p52 antibody, Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells, Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPM1 8226 a cell line involves upregulation of the p52 (NF-kappa B2) gene, which appears to be instrumental in the blockade of TNF gene expression

Mechanisms of hypoxic up-regulation of versican gene expression in macrophages

Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression

Influence of lung CT changes in chronic obstructive pulmonary disease (COPD) on the human lung microbiome

BACKGROUND: Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited. METHODS: Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons. RESULTS: We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype. CONCLUSION: Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system