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Human TRIM gene expression in response to interferons.

Carthagena L, Bergamaschi A, Luna JM, David A, Uchil PD, Margottin-Goguet F, Mothes W, Hazan U, Transy C, Pancino G, Nisole S - PLoS ONE (2009)

Bottom Line: We found that 27 of the 72 human TRIM genes are sensitive to IFN.Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity.Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.

View Article: PubMed Central - PubMed

Affiliation: Département des Maladies Infectieuses, Institut Cochin, Université Paris Descartes, CNRS, UMR 8104, Paris, France.

ABSTRACT

Background: Tripartite motif (TRIM) proteins constitute a family of proteins that share a conserved tripartite architecture. The recent discovery of the anti-HIV activity of TRIM5alpha in primate cells has stimulated much interest in the potential role of TRIM proteins in antiviral activities and innate immunity.

Principal findings: To test if TRIM genes are up-regulated during antiviral immune responses, we performed a systematic analysis of TRIM gene expression in human primary lymphocytes and monocyte-derived macrophages in response to interferons (IFNs, type I and II) or following FcgammaR-mediated activation of macrophages. We found that 27 of the 72 human TRIM genes are sensitive to IFN. Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity. Also, we found 2 TRIM proteins, TRIM9 and 54, to be specifically up-regulated in FcgammaR-activated macrophages.

Conclusions: Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.

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Related in: MedlinePlus

TRIM gene expression in human primary macrophages and lymphocytes.cDNA was prepared from primary macrophages (MDM) and lymphocytes (PBL) from 3 donors, as described in the Materials and Methods section. The expression of 86 genes, including 72 TRIM genes and 5 housekeeping genes, was analyzed by quantitative RT-PCR array. A. Comparison of the expression of 5 housekeeping genes in untreated MDM and PBL. The mean Ct values for each gene in untreated cells from 3 donors are shown. Error bars show standard deviation. RPL13A presented the smallest standard deviation values and was therefore selected for normalization. B. Constitutive expression of TRIM genes in MDM (M) and PBL (P). Histograms represent mean 2−ΔCt values for each gene±SD. C. Relative expression of TRIM genes in MDM (M) and PBL (P). Mean 2−ΔCt values were determined by subtracting RPL13A, and each sample was normalized to the median expression of each gene in both cell types. Resulting 2−ΔΔCt values were represented as a heat map, using Java TreeView. Green: low relative expression; Yellow: median value (same expression in MDM and PBL); Red: high relative expression.
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pone-0004894-g002: TRIM gene expression in human primary macrophages and lymphocytes.cDNA was prepared from primary macrophages (MDM) and lymphocytes (PBL) from 3 donors, as described in the Materials and Methods section. The expression of 86 genes, including 72 TRIM genes and 5 housekeeping genes, was analyzed by quantitative RT-PCR array. A. Comparison of the expression of 5 housekeeping genes in untreated MDM and PBL. The mean Ct values for each gene in untreated cells from 3 donors are shown. Error bars show standard deviation. RPL13A presented the smallest standard deviation values and was therefore selected for normalization. B. Constitutive expression of TRIM genes in MDM (M) and PBL (P). Histograms represent mean 2−ΔCt values for each gene±SD. C. Relative expression of TRIM genes in MDM (M) and PBL (P). Mean 2−ΔCt values were determined by subtracting RPL13A, and each sample was normalized to the median expression of each gene in both cell types. Resulting 2−ΔΔCt values were represented as a heat map, using Java TreeView. Green: low relative expression; Yellow: median value (same expression in MDM and PBL); Red: high relative expression.

Mentions: In order to perform a comprehensive study of TRIM gene expression in human primary lymphocytes and macrophages, unstimulated peripheral blood lymphocytes (PBL) and monocyte derived macrophages (MDM) from 3 donors were either left untreated or stimulated with type I IFN, type II IFN or immune complexes (IC, in the case of MDM only), as indicated in the Materials and Methods section. After RNA extraction and cDNA preparation, we screened 86 gene transcripts by real-time quantitative PCR. In addition to the 72 TRIM gene transcripts, we also analyzed the expression of a number of housekeeping genes to standardize the assays, such as PPIA (peptidylprolyl isomerase A, cyclophilin A), HPRT1 (hypoxanthine phosphoribosyltransferase 1), RPL13A (ribosomal protein L13a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ACTB (actin beta) (Table 1). We also included some genes whose expression is known to be either down-regulated by IFN, such as HIST4H4 (histone cluster 4, H4) or up-regulated, such as STAT1 (signal transducer and activator of transcription 1, 91kDa), EIF2AK2 (Homo sapiens eukaryotic translation initiation factor 2-alpha kinase 2, PKR), OAS2 (2′-5′-oligoadenylate synthetase 2, 69/71 kDa), MX1 (myxovirus resistance 1), ADAR (adenosine deaminase, RNA-specific), APOBEC3G and APOBEC3F (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G and 3F) (Table 1) [32], [46]. Furthermore, we analyzed CSF1 (or M-CSF, Macrophage colony-stimulating factor 1) expression as a control for IC-mediated MDM activation, since we have previously shown that CSF1 expression is up-regulated following FcγR cross-linking [39]. Finally, we included internal controls to account for genomic DNA contamination, reverse transcription efficiency and PCR efficiency, in order to validate the screen and allow proper comparison between experiments. All data were analyzed using the 2−ΔΔCt method. From the collected data, we first analyzed the basal expression of the screened genes in untreated MDM and PBL. In order to compare the basal expression of each gene in lymphocytes and MDM, we normalized our values by a housekeeping gene whose expression is as similar as possible in both cell types. Toward this end, we compared the mean Ct values for each housekeeping gene in untreated MDM and PBL. As shown in Figure 2A, RPL13A presents the smallest standard deviation values among the 5 selected housekeeping genes, demonstrating that its expression was almost identical in PBL and MDM. Therefore, we used RPL13A to compare TRIM gene expression between both cell types.


Human TRIM gene expression in response to interferons.

Carthagena L, Bergamaschi A, Luna JM, David A, Uchil PD, Margottin-Goguet F, Mothes W, Hazan U, Transy C, Pancino G, Nisole S - PLoS ONE (2009)

TRIM gene expression in human primary macrophages and lymphocytes.cDNA was prepared from primary macrophages (MDM) and lymphocytes (PBL) from 3 donors, as described in the Materials and Methods section. The expression of 86 genes, including 72 TRIM genes and 5 housekeeping genes, was analyzed by quantitative RT-PCR array. A. Comparison of the expression of 5 housekeeping genes in untreated MDM and PBL. The mean Ct values for each gene in untreated cells from 3 donors are shown. Error bars show standard deviation. RPL13A presented the smallest standard deviation values and was therefore selected for normalization. B. Constitutive expression of TRIM genes in MDM (M) and PBL (P). Histograms represent mean 2−ΔCt values for each gene±SD. C. Relative expression of TRIM genes in MDM (M) and PBL (P). Mean 2−ΔCt values were determined by subtracting RPL13A, and each sample was normalized to the median expression of each gene in both cell types. Resulting 2−ΔΔCt values were represented as a heat map, using Java TreeView. Green: low relative expression; Yellow: median value (same expression in MDM and PBL); Red: high relative expression.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654144&req=5

pone-0004894-g002: TRIM gene expression in human primary macrophages and lymphocytes.cDNA was prepared from primary macrophages (MDM) and lymphocytes (PBL) from 3 donors, as described in the Materials and Methods section. The expression of 86 genes, including 72 TRIM genes and 5 housekeeping genes, was analyzed by quantitative RT-PCR array. A. Comparison of the expression of 5 housekeeping genes in untreated MDM and PBL. The mean Ct values for each gene in untreated cells from 3 donors are shown. Error bars show standard deviation. RPL13A presented the smallest standard deviation values and was therefore selected for normalization. B. Constitutive expression of TRIM genes in MDM (M) and PBL (P). Histograms represent mean 2−ΔCt values for each gene±SD. C. Relative expression of TRIM genes in MDM (M) and PBL (P). Mean 2−ΔCt values were determined by subtracting RPL13A, and each sample was normalized to the median expression of each gene in both cell types. Resulting 2−ΔΔCt values were represented as a heat map, using Java TreeView. Green: low relative expression; Yellow: median value (same expression in MDM and PBL); Red: high relative expression.
Mentions: In order to perform a comprehensive study of TRIM gene expression in human primary lymphocytes and macrophages, unstimulated peripheral blood lymphocytes (PBL) and monocyte derived macrophages (MDM) from 3 donors were either left untreated or stimulated with type I IFN, type II IFN or immune complexes (IC, in the case of MDM only), as indicated in the Materials and Methods section. After RNA extraction and cDNA preparation, we screened 86 gene transcripts by real-time quantitative PCR. In addition to the 72 TRIM gene transcripts, we also analyzed the expression of a number of housekeeping genes to standardize the assays, such as PPIA (peptidylprolyl isomerase A, cyclophilin A), HPRT1 (hypoxanthine phosphoribosyltransferase 1), RPL13A (ribosomal protein L13a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ACTB (actin beta) (Table 1). We also included some genes whose expression is known to be either down-regulated by IFN, such as HIST4H4 (histone cluster 4, H4) or up-regulated, such as STAT1 (signal transducer and activator of transcription 1, 91kDa), EIF2AK2 (Homo sapiens eukaryotic translation initiation factor 2-alpha kinase 2, PKR), OAS2 (2′-5′-oligoadenylate synthetase 2, 69/71 kDa), MX1 (myxovirus resistance 1), ADAR (adenosine deaminase, RNA-specific), APOBEC3G and APOBEC3F (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G and 3F) (Table 1) [32], [46]. Furthermore, we analyzed CSF1 (or M-CSF, Macrophage colony-stimulating factor 1) expression as a control for IC-mediated MDM activation, since we have previously shown that CSF1 expression is up-regulated following FcγR cross-linking [39]. Finally, we included internal controls to account for genomic DNA contamination, reverse transcription efficiency and PCR efficiency, in order to validate the screen and allow proper comparison between experiments. All data were analyzed using the 2−ΔΔCt method. From the collected data, we first analyzed the basal expression of the screened genes in untreated MDM and PBL. In order to compare the basal expression of each gene in lymphocytes and MDM, we normalized our values by a housekeeping gene whose expression is as similar as possible in both cell types. Toward this end, we compared the mean Ct values for each housekeeping gene in untreated MDM and PBL. As shown in Figure 2A, RPL13A presents the smallest standard deviation values among the 5 selected housekeeping genes, demonstrating that its expression was almost identical in PBL and MDM. Therefore, we used RPL13A to compare TRIM gene expression between both cell types.

Bottom Line: We found that 27 of the 72 human TRIM genes are sensitive to IFN.Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity.Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.

View Article: PubMed Central - PubMed

Affiliation: Département des Maladies Infectieuses, Institut Cochin, Université Paris Descartes, CNRS, UMR 8104, Paris, France.

ABSTRACT

Background: Tripartite motif (TRIM) proteins constitute a family of proteins that share a conserved tripartite architecture. The recent discovery of the anti-HIV activity of TRIM5alpha in primate cells has stimulated much interest in the potential role of TRIM proteins in antiviral activities and innate immunity.

Principal findings: To test if TRIM genes are up-regulated during antiviral immune responses, we performed a systematic analysis of TRIM gene expression in human primary lymphocytes and monocyte-derived macrophages in response to interferons (IFNs, type I and II) or following FcgammaR-mediated activation of macrophages. We found that 27 of the 72 human TRIM genes are sensitive to IFN. Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity. Also, we found 2 TRIM proteins, TRIM9 and 54, to be specifically up-regulated in FcgammaR-activated macrophages.

Conclusions: Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.

Show MeSH
Related in: MedlinePlus