Oncogenic Merkel Cell Polyomavirus T Antigen Truncating Mutations are Mediated by APOBEC3 Activity in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer, which is frequently caused by Merkel cell polyomavirus (MCPyV). Mutations of MCPyV tumor (T) antigens are major pathologic events of virus-positive (MCPyV+) MCCs, but their source is unclear. Activation-induced cytidine deaminase (AID)/APOBEC family cytidine deaminases contribute to antiviral immunity by mutating viral genomes and are potential carcinogenic mutators. We studied the contribution of AID/APOBEC cytidine deaminases to MCPyV large T (LT) truncation events. The MCPyV LT area in MCCs was enriched with cytosine-targeting mutations, and a strong APOBEC3 mutation signature was observed in MCC sequences. AICDA and APOBEC3 expression were detected in the Finnish MCC sample cohort, and LT expression correlated with APOBEC3H and APOBEC3G. Marginal but statistically significant somatic hypermutation targeting activity was detected in the MCPyV regulatory region. Our results suggest that APOBEC3 cytidine deaminases are a plausible cause of the LT truncating mutations in MCPyV+ MCC, while the role of AID in MCC carcinogenesis is unlikely.</p

An insight into molecular mechanisms of human T helper cell differentiation.

Selective activation of T helper (Th) cell subsets plays an important role in immune response to pathogens as well as in the pathogenesis of human allergy and inflammatory diseases. Th1 cells along with the recently discovered Th17 cells play a role in the pathogenesis of autoimmune diseases. Th2 cytokines lead to series of inflammatory processes characteristic for asthma and other atopic diseases. To understand the pathogenesis of immune-mediated diseases it is crucial to dissect pathways and regulatory networks leading to the development of distinct Th subsets. Such knowledge may lead to better strategies for developing diagnostics and therapies for these diseases. The differentiation of Th1, Th2, and Th17 effector cells is driven by signals originating from T cell and costimulatory receptors as well as cytokines in the surroundings of activated naive T helper cells. There are several proteins involved in the regulation of this differentiation process. Most of the data on T helper cell differentiation have been acquired using mouse. In this review, we have summarized what is known about human T helper differentiation. In addition, selected differences between human and mouse will be discussed

Knockdown of c-FLIP_L affects apoptosis and proliferation of Th cells.

<p>Freshly isolated Thp cells were transfected with c-FLIP_S, c-FLIP_L or non-targeting (NT) siRNA, left to rest for 20–24 h and then activated and stimulated with IL-12 (Th1) or IL-4 (Th2). A. The knockdown efficiency of c-FLIP_S and c-FLIP_L siRNAs. Samples for western blotting analysis were harvested 24 h after priming. GAPDH was used as a loading control. Bars show relative levels of c-FLIP_L (upper panel) and c-FLIP_S (lower panel) obtained by quantifying and normalizing against the levels of GAPDH. The value of NT Th1 was set as 1. B. the knockdown of c-FLIP_L affects the proliferation of Th1 and Th2 cells. Transfected cells were left to rest for 20–24 h and then stained with CFSE and activated and stimulated with IL-12 (Th1) or IL-4 (Th2). The proliferation of CFSE stained cells was analyzed by flow cytometry at days 2, 3 and 4 after initiation of the culture. Histogram shows representative data of three independent biological replicate cultures at day 4. C. Bars show proliferative indexes calculated from three independent biological replicate cultures. Statistical significance was calculated using paired student's t-test, * p<0.05. D. Analysis of CD69 expression by flow cytometry at 24 h time-point. Results are calculated from three independent biological replicate cultures. E. c-FLIP_L knockdown Th cells have elevated levels of apoptotic cells. Transfected cells were left to rest for 24 h, cultured and activated in Th1 or Th2 conditions (as described above) followed by staining with Annexin-FITC and prodium iodide (PI). Representative data of three independent biological replicate cultures is shown. F. Bars represent the average of percentage of early apoptotic (Annexin-FITC+PI-) cells (±SEM). Results were calculated from three independent biological replicate cultures. Statistical significance was calculated using the paired student's t-test, * p<0.05. B–F. NT, cFLIP_S and cFLIP_L refer to the used siRNAs. SEM =  standard error of mean.</p

Induction of c-FLIP expression by TCR signaling and Th1/Th2 cytokines.

<p>Thp cells were isolated from cord blood and activated (Th0) or also stimulated with IL-12 (Th1) or IL-4 (Th2) and samples for real-time RT-PCR analysis were collected at indicated time-points. A–C. The expression of <i>c-FLIP_S</i> (A), <i>c-FLIP_L</i> (B) and <i>c-FLIP_R</i> (C) mRNA during early Th cell differentiation. The graph represents the fold change (±SEM) for Th0, Th1 and Th2 calculated against Thp-sample. A–C. Statistical significance was calculated using paired student's t-test (* p<0.05; Th2 vs Th0, # p<0.05; Th2 vs Th1 and ‡ p<0.05 Th1 vs Th0). Results are calculated from three independent biological replicate cultures. D. The expression levels of <i>c-FLIP_S</i> and <i>c-FLIP_R</i> mRNA measured by real-time RT-PCR were compared with each other and are represented as fold change (paired student's t-test, p<0.05; <i>c-FLIP_S</i> vs <i>c-FLIP_R</i> all time-points in Th0 (#), all time-points except 72 h in Th1 cells (‡); and all time-points except 2 h and 72 h in Th2 cells (*)). Results are calculated from three independent biological replicate cultures. SEM =  standard error of mean.</p

The Regulation and Role of c-FLIP in Human Th Cell Differentiation

<div><p>The early differentiation of T helper (Th) cells is a tightly controlled and finely balanced process, which involves several factors including cytokines, transcription factors and co-stimulatory molecules. Recent studies have shown that in addition to the regulation of apoptosis, caspase activity is also needed for Th cell proliferation and activation and it might play a role in Th cell differentiation. The isoforms of the cellular FLICE inhibitory protein (c-FLIP) are regulators of CASPASE-8 activity and the short isoform, c-FLIP_S, has been shown to be up-regulated by IL-4, the Th2 driving cytokine. In this work, we have studied the expression and functional role of three c-FLIP isoforms during the early Th cell differentiation. Only two of the isoforms, c-FLIP_S and c-FLIP_L, were detected at the protein level although c-FLIP_R was expressed at the mRNA level. The knockdown of c-FLIP_L led to enhanced Th1 differentiation and elevated IL-4 production by Th2 cells, whereas the knockdown of c-FLIP_S diminished GATA3 expression and IL-4 production by Th2 cells. In summary, our results provide new insight into the role of c-FLIP proteins in the early differentiation of human Th cells.</p></div

STAT6 is important for stable c-FLIP_S expression in Th2 cells.

<p>A. Thp cells were isolated from cord blood and activated (Th0) or also stimulated with IL-12 (Th1) or IL-4 (Th2) and samples for western blotting were collected at the indicated time-points. The panels show representative data from three independent biological replicate cultures. B. Bars represent the mean values (±SEM) of the relative levels of c-FLIP_S protein, obtained by quantifying and normalizing against the levels of β-ACTIN. The values of the Th0 samples were set as 1. Results were calculated from three independent biological replicate cultures. C. Freshly isolated Thp cells were transfected with STAT6 (S6) or non-targeting (NT) siRNA and polarized in Th0 or Th2 direction 20–24 h after transfection. Samples for western blotting were harvested at the indicated time-points. The panels show representative data of three biological replicate cultures. D. Bars represent the mean values (±SEM) of the relative levels of c-FLIP_L (upper panel) and c-FLIP_S (lower panel), obtained by quantifying and normalizing against the levels of GAPDH. The values of Th0 (24 h) and Th2 (24 h) were set as 1 and other Th0 and Th2 samples were compared to them. Results were calculated from three biological replicate cultures. Statistical significance was calculated using the paired student's t-test, * p>0.05. SEM =  standard error of mean.</p

The down-regulation of c-FLIP_L results in the increase of secreted IFNγ by human Th1 cells.

<p>Freshly isolated Thp cells were transfected and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102022#pone-0102022-g003" target="_blank">Figure 3</a>. Cell culture supernatants from Th1 polarized cells were collected at the indicated time-points, and the amount of secreted IFNγ produced by the cells was measured by cytokine assay. The bars represent the average secreted IFNγ in pg/ml (±SEM). Data is average of 5–6 independent cultures depending on time-point. Statistical significances were calculated using the paired student's t-test, *p<0.05. NT =  non-targeting, cFLIP_S and cFLIP_L refer to the used siRNAs. SEM =  standard error of mean.</p

Knockdown of c-FLIP influences the cytokine production of Th1 and Th2 cells.

<p>Cells were cultured as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102022#pone-0102022-g003" target="_blank">Figure 3</a>. A. c-FLIP_S, c-FLIP_L or non-targeting (NT) siRNA transfected cells were cultured in Th1 and Th2 polarizing conditions for 7 days. The cells were then harvested and restimulated or left unstimulated (control) and the levels of intracellular IFNγ and IL-4 were analyzed by flow cytometry. Dot plots show representative data of at least 7 independent biological replicate cultures. B. Bars represent the average percentage of IFNγ+ cells (±SEM) calculated from 7 independent cultures. C. Bars represent the average percentage of IL-4+ cells (±SEM) calculated from 8 independent cultures. B and C. Statistical significances were calculated using the paired student's t-test, *p<0.05. NT, cFLIP_S and cFLIP_L refer to the used siRNAs. SEM =  standard error of mean.</p

Knockdown of c-FLIP affects Th1 and Th2 markers.

<p>Freshly isolated Thp cells were transfected and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102022#pone-0102022-g003" target="_blank">Figure 3</a>. Samples for real-time RT-PCR analysis were collected at indicated time-points. A. <i>IL12Rβ2</i> mRNA levels of transfected Th1 cells were analyzed. The graph shows average fold differences (±SEM) in the siRNA treated Th1 cells compared with Thp sample. The data is calculated from 3 independent cultures. B. <i>TBET</i> mRNA levels of transfected Th1 cells were analyzed. The graph shows average fold differences (±SEM) in the siRNA treated Th1 cells compared with Thp sample. The data is calculated from 5 independent cultures. C. <i>IFNG</i> mRNA levels of transfected Th1 cells were analyzed. The graph shows average fold differences (±SEM) in the c-FLIP_S and c-FLIP_L siRNA treated Th1 cells compared with non-targeting (NT) siRNA treated Th1 cells. The data is calculated from 4 independent cultures. D. <i>GATA3</i> mRNA levels of transfected Th2 cells were analyzed. The graph shows average fold differences (±SEM) in the siRNA treated Th2 cells compared with Thp sample. The data is calculated from 5 independent cultures. Statistical significances were calculated using the paired student's t-test, * p<0.05; ** p<0.01. SEM =  standard error of mean.</p

Genes and pathways revealed by whole transcriptome analysis of milk derived bovine mammary epithelial cells after Escherichia coli challenge

Abstract Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (Padj < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway