Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. Inhaled PM induces innate immune responses by airway epithelial cells that may lead to the exacerbation or de novo development of airway disease. We have previously shown that 10-μm PM (PM10) activates the nucleotide-binding domain, leucine-rich repeat protein (NLRP) 3 inflammasome in human airway epithelial cells. Our objective was to determine the innate and adaptive immune responses mediated by the airway epithelium NLRP3 inflammasome in response to PM10 exposure. Using in vitro cultures of human airway epithelial cells and in vivo studies with wild-type and Nlrp3(-/-) mice, we investigated the downstream consequences of PM10-induced NLPR3 inflammasome activation on cytokine production, cellular inflammation, dendritic cell activation, and PM10-facilitated allergic sensitization. PM10 activates an NLRP3 inflammasome/IL-1 receptor I (IL-1RI) axis in airway epithelial cells, resulting in IL-1β, CC chemokine ligand-20, and granulocyte/macrophage colony-stimulating factor production, which is associated with dendritic cell activation and lung neutrophilia. Despite these profound innate immune responses in the airway epithelium, the NLRP3 inflammasome/IL-1RI axis is dispensable for PM10-facilitated allergic sensitization. We demonstrate the importance of the lung NLRP3 inflammasome in mediating PM10 exposure-associated innate, but not adaptive, immune responses. Our study highlights a mechanism by which PM10 exposure can contribute to the exacerbation of airway disease, but not PM10-facilitated allergic sensitization.

Key Points• A functional demonstration of the oncogenic role of mutated EZH2 in a mouse model is presented.• The global effects of mutated EZH2 on expression and epigenome have been characterized.The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2 Y641F is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2 Y641F by crossing these transgenic mice with Em-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2 Y641F. The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2 Y641F can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments. (Blood. 2014;123(25):3914-3924)

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Background Clinical trial samples may be stored frozen for prolonged periods before analysis, which can reduce the immunoreactivity of numerous analytes, particularly peptides. We sought to determine the effect of 6 years of frozen storage on serum N-terminal pro–B-type natriuretic peptide (NT-proBNP). Methods NT-proBNP was measured from serum samples taken from 99 different patients enrolled in the CanPREDDICT study after <1 year of storage at −70 °C using the Roche first-generation NT-proBNP assay on an e411 instrument. Separate aliquots of the same samples were analyzed after an additional 6 years of storage at −70 °C using the Roche second-generation assay on an e601 instrument. Results The median NT-proBNP immunoreactivity for the first measurement was 572 pg/mL (interquartile range [IQR] 205–1606, range 49–12820), while after an additional 6 years of storage at −70 °C, this value decreased to 526 pg/mL (IQR 181–1338, range 18–12880), resulting in a median percent difference of −7% (IQR −10.6% to −3.4%, P < 0.001). Conclusions We report findings consistent with trends seen in previous work but have investigated the effect of a much longer storage period. Larger percent decreases in NT-proBNP reaching statistical significance are seen, although the median difference is still <10%.

The histone methyl-transferase EZH2 is frequently affected by gain-of-function mutations in germinal-center lymphoma. To further test if these EZH2 mutations can be driver mutations in lymphomagenesis, we have previously generated and characterized a mouse line transgenic for EZH2Y641F. This mouse model leads to an acceleration of lymphoma development in combination with Myc. We have now further investigated this model system and have used RNAseq and ChIPseq to characterize the effects of mutated EZH2 on gene expression and the epigenome. We have previously shown that in contrast to lymphomas observed with Emu-Myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2Y641F, present with a B220+IgM+phenotype. We have now characterized the accumulating B cell subset by performing in-depth immunophenotyping. This analysis showed that the cells are IgDlo, CD21-, CD23- and partially express AA4.1. This marker combination is consistent with the transitional stage (T1) of B cell development. For the global gene expression and histone methylation analysis, splenic B cells (B220+) were isolated from one Eμ-Myc and one Eμ-Myc/EZH2Y641F mouse before the onset of disease symptoms. The samples were then processed for RNA sequencing and ChIP sequencing. ChIP was performed using validated antibodies for H3K4me3 and H3K27me3. Of the 22,137 genes studied we observed that 1,112 (412) genes were down-regulated and 788 (209) genes were up-regulated in Eμ-Myc/EZH2Y641F mice with p-value<0.05 (FDR<0.1) when only genes with RPKM > 0.05 and a minimum number of reads of 30 were considered. An integrated analysis of the RNA sequencing data with the ChIP sequencing data for H3K27me3 and H3K4me3 was performed, and indicated that the altered epigenome of the Eμ-Myc/EZH2Y641F mouse impacted protein-coding gene expression. This analysis showed that genes down-regulated in the Eμ-Myc/EZH2Y641F mouse have increased H3K27me3 marks at their transcription start site indicative of a significant fraction of these genes being regulated by this mark. In contrast, genes up-regulated in the Eμ-Myc/EZH2Y641Fmouse mainly exhibited increased H3K4Me3 marks at their transcription start site. We then used DAVID/KEGG and the MGSA package to identify pathways associated with mutated EZH2 and identified GO terms that were enriched by the differentially expressed genes. Interestingly this analysis returned many important pathways in B cell regulation and immune function. The identified pathways include the B cell receptor signalling pathway and the JAK-STAT signaling pathway. We previously observed an increase in the proliferation rate of splenic B cells in Eμ-Myc/EZH2Y641Fmice. While cell-cycle genes were not specifically enriched, there were several of these genes that were found deregulated (including e. g. CyclinD1). In summary, we were able to identify several key pathways that may be contributing to the acceleration of lymphoma development observed with EZH2Y641F and may also be important for the understanding of pathogenesis of EZH2 mutated lymphoma. Disclosures: No relevant conflicts of interest to declare.