Immature monocyte derived dendritic cells gene expression profile in response to Virus-Like Particles stimulation

Abstract

We have recently developed a candidate HIV-1 vaccine model based on HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from an Ugandan HIV-1 isolate of the clade A (HIV-VLPAs). The HIV-VLPAs induce in Balb/c mice systemic and mucosal neutralizing Antibodies as well as cytotoxic T lymphocytes, by intra-peritoneal as well as intra-nasal administration. Moreover, we have recently shown that the baculovirus-expressed HIV-VLPs induce maturation and activation of monocyte-derived dendritic cells (MDDCs) which, in turn, produce Th1- and Th2-specific cytokines and stimulate in vitro a primary and secondary response in autologous CD4+ T cells. In the present manuscript, the effects of the baculovirus-expressed HIV-VLPAs on the genomic transcriptional profile of MDDCs obtained from normal healthy donors have been evaluated. The HIV-VLPA stimulation, compared to both PBS and LPS treatment, modulate the expression of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation. The results of gene profiling analysis here presented are highly informative on the global pattern of gene expression alteration underlying the activation of MDDCs by HIV-VLPAs at the early stages of the immune response and may be extremely helpful for the identification of exclusive activation markers.

Figure 1

Maturation of DCs by baculovirus-expressed HIV-VLPs. Immature MDDCs were incubated in the presence of the indicated stimulus for 16 hours. The expression of CD80, CD83, CD86 and HLA-DR was analyzed on fixed cells by FACScalibur flow cytometer and data analysis was carried out with FlowJo software. The results of a representative experiment are shown; the shadowed curve represents the untreated cells.

Figure 2

Unsupervised hierarchical clustering of all filtered data. The clusterogram represents 3,119 genes obtained by Eisen hierarchical clustering of the complete 17K dataset filtered for genes that are expressed in a minimum of 80% of the samples 4 and 8 h after HIV-VLPs or LPS stimulation. The PBS treatments was evaluated after 8 h of stimulation. The clustering is defined by the dendrogram and each treatment/time point is represented by a single branch.

Figure 3

Pattern of gene expression in human monocyte-derived DCs. Regulated genes by HIV-VLPs or LPS treatment in MDDCs, showing at least a 2-fold modulation (up or downregulation), have been evaluated. Each circle represents the whole set of genes identified in the indicated comparisons, based on the 2-fold modulation parameter. Numbers in overlapping regions represent common regulated genes. Numbers in non-overlapping regions represent unique regulated genes. Circles are drawn in arbitrary scale.

Figure 4

Supervised hierarchical clustering of genes differentially expressed in HIV-VLP-treated MDDCs. The clusterograms represent an Eisen hierarchical clustering of the 281 genes differentially expressed (p < 0.005) in HIV-VLPs-treated MDDCs, compared to PBS treatment (A), and of 217 genes differentially expressed (p < 0.005) in HIV-VLPs-treated MDDCs, compared to LPS treatment (B). The clustering is defined by the dendrogram on the top and on the side of the clusterogram.

Figure 5

Supervised hierarchical clustering of genes upregulated by HIV-VLPs in MDDCs. 3,119 genes included in this analysis were filtered upon the criteria of showing less than 20% missing data and a minimum of 3-fold modulation in expression. The expanded section shows nodes including genes upregulated by HIV-VLPs. Individual genes are indicated on the right.

Figure 6

Supervised hierarchical clustering of genes upregulated by HIV-VLPs and LPS in MDDCs. 3,119 genes included in this analysis were filtered upon the criteria of showing less than 20% missing data and a minimum of 3-fold modulation in expression. The expanded section shows nodes including genes upregulated by HIV-VLPs and LPS-4 h (top) or by HIV-VLPs and LPS (bottom). Individual genes are indicated on the right.

Figure 7

Supervised hierarchical clustering of genes downregulated by HIV-VLPs and LPS in MDDCs. 3,119 genes included in this analysis were filtered upon the criteria of showing less than 20% missing data and a minimum of 3-fold modulation in expression. The expanded section shows nodes including genes downregulated by HIV-VLPs only or by HIV-VLPs and LPS-4 h are shown (top and bottom, respectively). Individual genes are indicated on the right.