Characterization of early host responses in adults with dengue disease

Dengue virus (DENV) is endemic throughout the tropics and in many subtropical parts of the world, causing significant human morbidity and mortality [1]. An understanding of the host response, as obtained from genome-wide transcriptional profiling of dengue infection may reveal unique patterns pertaining to specific disease outcomes and identify molecular mechanisms that could be targeted pharmacologically [2, 3] and a number of studies of the host transcriptional responses to DENV infection have been performed. Comparing the results from in-vitro DENV-infected cells; HUVEC [4], non-small lung cancer cells [5], HepG2 [6, 7]; primary human immune cells [8] and muscle satellite cells [9]; to whole blood or isolated mononuclear cells from DENV-infected patients [10–14], the first studies reports abundant transcripts related to innate immunity while the latter largely report non-immune related transcripts of ER-stress, oxidative metabolism and signal transduction. However, the human studies sampled patients at the time of hospital admission or after 4-5 days of illness, focusing on differences between clinical phenotypes. We hypothesized that earlier assessment following symptom presentation would be required to characterize the in-vivo dengue innate immune response and that the early host responses may reflect components of the disease pathogenesis.

Taken together, at this previously unassessed early stage of dengue disease the innate immune responses predominate, with the most significant canonical pathways being IFN-signaling, pattern recognition signaling and complement activation, both in relation to autologous transcripts in convalescence and to whole blood transcripts in non-dengue febrile illnesses at a similar phase following fever onset. In the convalescent phase of dengue disease, pathways related to adaptive immune responses are active, rendering these genes to appear down-regulated at the acute phase and in concordance with previous reports on transcripts derived from dengue disease in or just before defervescence [10–14], non-immune canonical pathways dominate. Interestingly, many of the responses seen in acute dengue in relation to the convalescent baseline were replicated when comparing to other non-dengue febrile illnesses, indicating a more prominent IFN-response, specific to dengue disease, and a selective utlization of TLR7, MDA5 and OAS. We do not have information of the etiology of the non-dengue febrile controls, but etiological search in patients enrolled later in the same cohort have shown that viral infections such as influenza, adenovirus and metapneumovirus are common among the DENV RT-PCR negative controls. IFN-responses arise hours after viremia is established and are likely of great importance for the control of viral replication. The IFN-response in dengue infection have been shown to be activated through two main pathogen recognition families; the TLRs and the RLRs. Of the TLRs, TLR7 in dendritic cells has been shown to interact with DENV RNA leading to viral fusion and uncoating processing that in the end activated a type-1 IFN-response [24]. TLR3 was also shown to have a role in the regulation of the inflammatory response in dengue infected umbilical vein endothelial cells [4]. When we compared the acute samples with the samples collected at convalescence in our dengue positive patients we saw an up regulation of TLR7 and IRF7 which indicates an activation of the TLR7 signalling pathway. TLR7 is an intracellular receptor that senses microbial nucleic acids and via IRF7 induces a strong type-1 IFN-response, particularly IFNα [25]; while it can also induced a type-1 IFN-response via NFκB [26]. RLR activation was also seen when comparing acute versus convalecent samples in the DENV RT-PCR positive patients, with the genes RIG1 and MDA5 upregulated. A recent study from Fredericksen et al demonstrated that RIG1 and MDA5 induce an IFN-response in West Nile virus-infected fibroblasts by activation of IRF3 [27]. They also show that RIG1 primes an early IFN-response while MDA5 is more involved in the second phase of IFN-dependent gene expression. Both RIG1 and MDA5 have also been shown to be activated in double RIG1/MDA5 knockout mouse fibroblasts [28, 29]. Interestingly, RIPK2 (RIP2) was also identified as an over expressed gene indicating activation of the NLR signalling pathway. NOD1 and NOD2, members of the NLR protein family, are activated by specific bacterial peptides, and via RIPK2 induce NFκB activation [30]. There are no earlier reports on virus induced NLR activation. Transcripts clustering to the PPAR pathway were also among the most significantly enriched comparing dengue to non-dengue. PPARγ has been shown to play a critical role in the control of adipocyte differentiation and lipid metabolism, and also immunity and the barrier functions of epithelial and endothelial cells; in dengue disease this could be a response to epithelial stress [31, 32]. Furthermore, among the most highly enriched gene transcripts is JUP (plakoglobin), that encode a protein that forms part of desmosomes and intermediate junctions in endothelial cells, wich could indicate that some transcripts were derived from affected endothelial cells. In fact, high numbers of detached endothelial cells in peripheral blood has been obseved in the acute phase of dengue [33]. DEFB1 was another highly enriched transcript which encodes an antimicrobial peptide, β- defensin 1, that has mostly been studied in the context of epithelial protection against HIV [34]. Also among the highly enriched transcripts were genes of the secreted mediators CCL2, CCL8, CXCL10 and CCL3, whose proteins were also found to be adundant in patient serum (Table 2). These have been observed before in dengue disease and MCP-1 could well participate in the pathogenesis of vascular leakage by its effect on endothelial tight junctions [35, 36]. Other genes that were significantly enriched but did not cluster to any canonical pathway were NMES1 and CCNA1. While CCNA1 is a cyclin which probably mediates cell cycle arrest to prevent virus replication in infected cells, little is known about the gene NMES1. A recent study has identified that the transcript is a primary functional microRNA (miR-147) which was expressed in murine macrophages upon TLR-stimulation [37]. It was shown to negatively regulate inflammatory cytokine expression in these cells. As dengue virus is believed to primarily replicate in cells of monocyte/macrophage lineage, NMES1 expression may be important to moderate the pro-inflammatory cytokine release from these cells, especially as these are potential mediators of pathology. Comparing dengue patients with different DENV-serostatus at inclusion rendered a surprisingly small number of differentially expressed genes with no significant pathway clustering. On the individual gene level PRDX2, an antioxidant enzyme was enriched in the acute stage of secondary DENV infection, while at defervesence, CCR2 was among the transcripts seen upregulated in secondary infection. Sierra et al. addressed this issue by examining in-vitro infected PBMC from immune and non-immune individuals and showed expression of CCL2 to be highly dependent on previously infecting serotype at 24 h post infection [38]. CCL2 was not an found an enriched transcript in acute secondary infection in our material but the number of patients in each group in this specific comparison was small (ten patients were DENV RT-PCR and DENV-IgG positive) and information of previous infecting serotype was lacking, thus this specific assessment should be perfomed in a larger cohort to allow further speculation on these genes in the context of dengue pathogenesis.

In summary, early DENV-induced transcriptional host responses in-vivo are predominatly involving innate immune responses and overlaps to a large extent with those described in in-vitro, where early transcriptional assessment post-infection has been easily obtained. Several of the early transcripts identified deserve further assessment in correlation to severe disease.