Dengue NS1 interaction with lipids alters its pathogenic effects on monocyte derived macrophages

Abstract

Background: While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there have been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity.

Methods: Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF=8) or dengue haemorrhagic fever (DHF=8). IL-1β levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF.

Results: Significantly higher levels of IL-1β were seen in culture supernatants when NS1 was co-cultured with LDL (p=0.01), but with lower levels with HDL (p=0.05). MDMs of those past DHF produced more IL-1β when NS1 with PAF (p=0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF.

Conclusions: As RPLP2 is important in DENV replication and in regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

Figure 1.. IL-1β levels in culture supernatant of primary human monocyte derived macrophages (MDMs) co-cultures with NS1 and different lipid mediators.

MDMs from individuals with a history of DF (n=8) and DHF (n=8) were co-cultured with NS1 alone, different lipid mediators alone or with NS1 and different lipid mediators and the IL-1β levels in the supernatants were measured by ELISA after 24hrs. The Wilcoxon matched pairs signed rank test was used for comparison of IL-1 β levels in culture supernatants in the same individual under different conditions (A). IL-1β levels in culture supernatant in those with past DF vs DHF, under different culture conditions were compared using Mann-Whitney U test for unpaired groups (B). The lines indicate the median and IQR.

Figure 2.. Patterns of expression of different genes in monocyte derived macrophages (MDMs) when co-cultured with NS1 and different lipid mediators.

A heat-map was generated for expression of RIGI, IL-10, NLRP3, IFN-β, SMPD1, RPLP2, when MDMs were co-cultured from individuals with a history of DF (n=6) and DHF (n=6) with NS1 alone, different lipid mediators alone or with NS1 and different lipid mediators. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters under different culture conditions of NS1 with different lipid mediators. The color intensity corresponds to the Z-score that indicates the number of standard deviations by which the normalized raw value was below or above. The differences of monocyte subpopulations identified of each individual is indicated in the Y axis.

Figure 3.. Differences in gene expression patterns in monocyte derived macrophages (MDMs) when co-cultured with NS1 and different lipid mediators in those with past DF and DHF.

A volcano plot was generated to identify significant differences in gene expression in MDMs in those with past DF (n=6) and past DHF (n=6), with NS1 alone, different lipid mediators alone or with NS1 and different lipid mediators (A). Significant differences in RPLP2 gene expression were observed when MDM2s of those with past DHF were co-cultured with PAF, NS1 and PAF and NS1 and LDL, which are also displayed in a 3D plot view with individuals with DHF (blue) and DF (red) (B). A heatmap was generated for differential expression of RPLP2 identified by the volcano plot in those with past DF and DHF. The differences in monocyte subpopulations identified of each individual is indicated in the Y axis (C).