Blockade of T cell contact-activation of human monocytes by high-density lipoproteins reveals a new pattern of cytokine and inflammatory genes

Abstract

Background: Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact.

Methodology/principal findings: To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNgamma, CCL2, CCL5, TNF, IL-1beta, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1beta, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E(2)-both involved in inflammatory response-were triggered by T cell contact and inhibited in the presence of HDL.

Conclusions/significance: These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.

Figure 1. Apo A–I mediates the inhibitory effects of HDL in T cell contact-induced cytokine production in monocytes.

Monocytes (5×10⁴ cells/200 µl/well; 96-well plates) were activated (closed symbols) or not (open circles) by CE_sHUT for 24 h in the presence (triangles) or absence (circles) of HDL. Cell culture supernatants were tested by Quantikine kits (R&D) for the presence of IL-1β and TNF. Results are expressed as mean±SD of a representative experiment carried out in triplicate.

Figure 2. Modulation of cytokine production by HDL in CE_sHUT-activated monocytes.

Monocytes (5×10⁴ cells/200 µl/well; 96-well plates) were activated by CE_sHUT for 24 h in the presence (empty columns) or absence (grey columns) of HDL. Cell culture supernatants were tested for the presence of the indicated cytokines by BioPlex using a 27-Plex kit. Results were obtained from monocytes isolated from 3 individual donors and are expressed as mean ± SD (n = 3). (A) Cytokines previously shown to be modulated by CE_sHUT; (B) CE_sHUT-induced cytokines whose expression was not modulated by HDL; and (C) CE_sHUT-induced cytokines whose expression was inhibited in the presence of HDL.

Figure 3. Inhibition by HDL of CE_sHUT-triggered IL-1β mRNA.

Monocytes (7×10⁶ cells/P-100mm/6 ml) isolated from 3 different donors (a, b and c) were activated by CE_sHUT (6 µg/ml) in the presence (empty columns) or absence (grey columns) of HDL (0.2 mg/ml) for 3 h. (A) Isolated RNA was subjected to qPCR to assess IL-1β transcript levels. Results are presented as mean ± SD of triplicates. (B) Isolated RNA was subjected to microarray analysis. Results are expressed as mean ± SD, n = 3.

Figure 4. Analysis of gene expression in monocytes activated by CE_sHUT.

Total RNA was used in Affymetrix microarrays to analyse gene expression. The scatter plot (GeneSpring) shows the amounts expressed in CE_sHUT-activated monocytes (y-axis) as a function of amounts expressed in resting monocytes (x-axis), applying a cut-off of 2.0. Each dot represents one probe set; probe sets for IL-1β, IL-1Ra and TNF are indicated. Probe sets with signals below 100 were discarded (dashed lines). Using the latter criteria, 437 probe sets were up-regulated, and 229 down-regulated as indicated.

Figure 5. qPCR assessment of selected transcripts to validate microarray results.

Several genes listed in Table S4 were selected and RNA, isolated from the 3 monocyte preparations, was subjected to qPCR. (A) Genes that were validated (i.e., those displaying similar behavior as in microarray analysis). (B) Genes that were not validated. Results were normalized to fully activated condition (CE_sHUT), considered to be 1. Data are expressed as mean ± SD, n = 3; ** p<0.01; * p<0.05.

Figure 6. HDL inhibit miR-155 expression and PGE₂ production induced by CE_sHUT in human monocytes.

(A) Total RNA isolated from monocytes activated (CE_sHUT) or not (medium) in the presence (white columns) or absence (grey columns) of HDL and subjected to qPCR analysis for the presence of miR-155. (B) Monocytes were activated as in Figure 2, and PGE₂ production measured in cell supernatants. Results are expressed as mean ± SD of 3 different experiments.

Figure 7. HDL inhibit inflammatory functions induced by CE_sHUT in human monocytes.

Microarray results of [CE_sHUT] versus medium (grey columns) and [CE_sHUT + HDL] versus medium (empty columns) were subjected to comparative analysis in the IPA system. (A) Genes related to “Diseases and Disorders”; (B) genes related to “Molecular and Cellular Functions”.