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Characterization of the placental macrophage secretome: implications for antiviral activity.

ABSTRACT: It is well documented that placental macrophages show lower levels of HIV-1 infection than monocyte-derived macrophages (MDM). We used proteomic methods to test the hypothesis that placental macrophages secrete different proteins as compared to MDM that may contribute to decreased HIV-1 replication. Placental macrophages and MDM were cultured for 12 days and supernatant was collected. To characterize supernatants, the protein profiles of placental macrophages and MDM were compared using the protein chip assay. Subsequently, proteins were separated by one-dimensional gel electrophoresis and identified by tandem mass spectrometry at the corresponding mass to charge (m/z) range of 5000-20,000. Significant differences were found between placental macrophages and MDM in seven protein peaks with m/z values of 6075, 6227, 11,662, 14,547, 6158, 7740, and 11,934 on the CM10 and IMAC chips. After sequencing and identification, five proteins were validated for differential expression in placental macrophages and MDM by Western blot analyses. Peroxiredoxin 5, found to be more abundant in placental macrophage supernatants, is important in the cellular antioxidant mechanisms, and other members of its family have shown antiviral activity. Cystatin B was less abundant in PM supernatant, and decreased intracellular levels have recently been shown to be associated with lower HIV-1 replication in placental macrophages than in MDM. This study elucidates for the first time the placental macrophage secretome corresponding to 5000-20,000 Da and advances our understanding of the proteins secreted in the placenta that can protect the fetus against HIV-1 and other viral infections.

SUBMITTER: Garcia K

PROVIDER: S-EPMC2947718 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Characterization of the placental macrophage secretome: implications for antiviral activity.

García K K García V V Pérez Laspiur J J Duan F F Meléndez L M LM

Placenta 20081212 2

It is well documented that placental macrophages show lower levels of HIV-1 infection than monocyte-derived macrophages (MDM). We used proteomic methods to test the hypothesis that placental macrophages secrete different proteins as compared to MDM that may contribute to decreased HIV-1 replication. Placental macrophages and MDM were cultured for 12 days and supernatant was collected. To characterize supernatants, the protein profiles of placental macrophages and MDM were compared using the prot ...[more]

PMID: 19070362

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Project description:A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-alpha AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-alpha. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-alpha AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-alpha. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products. Gene expression was initially measured in four Daudi cell groups (3 x 10(6) in 10 ml of RPMI) treated for 24 h at IFN concentrations selected to allow comparison of gene activity in AV activity environments with environments in which no AV activity was observed. These treatment groups were: (i) 0.0036 ng of IFN-alpha2c/ml (n=5) (allowing AV activity only); (ii) 0.00036 ng of IFN-alpha2c/ml (n=5) (no AV observed); (iii) 0.036 ng of HY-2/ml (n=3) (allowing AV activity); and (iv) 0.0036 ng of HY-2/ml (n=3) (no AV observed). To refine the list of genes associated with AV activity, samples showing AP phenotype (achieved by treatment with IFN-alpha2c [0.36 ng/ml] (n=3) or HY-2 [36 ng/ml]) (n=3) were compared to identically treated samples manipulated to display the AV phenotype through subsequent neutralization with anti-IFNAR1 MAb 64.10 (1 ug/ml) (n=6). Further analysis of gene expression profiles after 6 h of incubation helped indicate genes associated with early roles in IFN-induced AV mechanisms (n=6).

Dataset Information

Characterization of the placental macrophage secretome: implications for antiviral activity.

Publications

Characterization of the placental macrophage secretome: implications for antiviral activity.

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