The role of lipids in the inception, maintenance and complications of dengue virus infection

Dengue fever is a viral condition that has become a recurrent issue for public health in tropical countries, common endemic areas. Although viral structure and composition have been widely studied, the infection phenotype in terms of small molecules remains poorly established. This contribution providing a comprehensive overview of the metabolic implications of the virus-host interaction using a lipidomic-based approach through direct-infusion high-resolution mass spectrometry. Our results provide further evidence that lipids are part of both the immune response upon Dengue virus infection and viral infection maintenance mechanism in the organism. Furthermore, the species described herein provide evidence that such lipids may be part of the mechanism that leads to blood-related complications such as hemorrhagic fever, the severe form of the disease.

study used no chromatographic approach. We intended to analyze samples with the least possible preparation and manipulation, attempting to minimize as much as possible changes in the biological matrix used. Additionally, direct infusion high-resolution mass spectrometry allows us to analyze a wide range of lipids, a characteristic that is impaired when using LC-MS, since the column separates lipids by their respective physicochemical characteristics such as polarity, isomerism and others 12,13 .

Metabolomic approach of serum of patients infected with DENV-4
PCA clearly shows the separation between Control and DENV-infected patients, as shown in Fig. 1. This multivariate data analysis method was chosen because it is an unsupervised approach, capable of reducing the number of variables (reduction of dimensions) in the original dataset (raw data) based on the similar features between the samples, helping to find the most representative variables (features) responsible for each of the two clusters formed 14 , according to Fig. 1. This enabled the election of a feature set that is characteristic for each analyzed group, namely DENV-infected patients and healthy individuals. The bidimensional score plot in Fig. 1 is derived from the analysis performed with the data collected by mass spectrometry in the positive mode; from this clustering, we selected and characterized the features (potential metabolomic markers) that presented the greatest relevance in discriminating both groups. To illustrate the characteristic markers chosen by PCA, a heatmap of all features selected by this model was built using Pearson's distance measurement and Ward's clustering algorithm (Fig. 2).The Fig. 2 clearly illustrates the differences in the pool of metabolites between both analyzed groups. Following structure elucidations by mass accuracy and MS/MS reactions 15 Table 1. Moreover, by elucidating relevant species such as the above mentioned, this work has confirmed that it is possible to identify in vivo which are the lipids associated with the phenotype of viral infection process by directly infusing the serum of infected patients in an HRMS instrument, regardless of any previous chromatographic approach.

Compounds involved in the pathophysiological mechanism of DENV-4 infection
Our findings corroborate previous reports by other groups, in studies that identified the production of a series of polypeptides that act as inflammatory mediators during the immune response of the host 3 , participating in the autophagy process by signaling between virus and host cell 2 and in their replication process 16 . These studies have also reported the synthesis of lipid mediators during viral infection, which are related to the signaling, control, and maintenance of both the immune response and DENV pathogenesis 6,[17][18][19] .

Phosphatidylcholines (PC) and Triglycerides (TG)
The importance of lipid changes during DENV cell infection is evident when the virus assumes control of the cellular metabolism by controlling and regulating autophagy mechanisms to meet the needs of the viral replication 20 .   Autophagy is a general term used to refer to pathways by which cytoplasmic material (soluble macromolecules and organelles) are delivered to the lysosomes for degradation 21 . There are three different processes characterized by autophagy: macroautophagy, autophagy mediated by chaperones, and microautophagy 21 . By these processes, an eukaryotic cell is able to promote essential lysosomal degradation for survival, differentiation, development and homeostasis, presenting an important adaptive role in the protection of organisms against several pathologies 22 . Eliminating defective proteins and organelles with the potential to trigger pathogenic processes prevents the abnormal accumulation of protein aggregates and the removal of obligate intracellular parasites (OIPs); additionally, the autophagic process also plays an important role in the innate and adaptive immunity: it is responsible for the formation of epitopes presented by MHC complexes 23 . Autophagy is rapidly and positively regulated by cells that need to obtain intracellular nutrients, either during a period of nutrient deprivation or absence of growth factors, as well when there is high energy demand 22 . In this way, DENV, like any other virus, controls the cellular mechanisms in its favor 20 . By taking control of the autophagy processes, the virus is able to control cellular lipid metabolism 24 , providing the demands required in the viral infection process 20 .
Viral growth occurs through the formation of viral replication complexes (VRCs) 25 , consisting of lipid vesicles constructed by all positive-strand RNA viruses from the reorganization of the host intracellular membranes; within this vesicle, viral assembly occurs 26 . Zhang, Jiantao, et al. (2016) demonstrated that a significant increase in PC is associated with viral replication, and occurs mainly in the perinuclear membrane of the endoplasmic reticulum (ER), where viral replication occurs; additionally, their data showed that PC accumulation is due to the formation of this lipid class at the region where the VRC will remain, and not due to the transport of preexisting PC in the cellular interior 25 . Thus, the 3 PCs identified herein as characteristic molecules for the group of infected patients (Table 1) are putatively related to the pool of PCs synthesized by the infected cells for viral replication. These data are not only in agreement with the role of PCs during viral infection by positive-chain RNA viruses 6,11,25 , but also demonstrate that the results obtained by direct in vitro analysis are corroborated in vivo.
Since DENV controls the lipid metabolism of the host cell 20,24 , and there is a urge for an additional bioenergetic demand in the viral replication process, the virus promotes the mobilization and recruitment of lipid droplets responsible for the cellular stock of TGs and cholesteryl esters 27 . Recruited TGs undergo the action of lipases in order to provide the necessary fatty acids (FA) for the additional energy supply, since these TGs are used for the production of ATP through the β-oxidation pathway 28 . Cholesterol, also released during this process, will be used to form VRC for viral replication, as well as PCs [29][30][31] . Thus, TGs identified in this study (Table 1) were indicated as markers present in serum of patients infected with DENV, since the increased bioenergetic demand for viral replication leads to a higher recruitment of TGs as an energy source 24 .

The role of platelet activation factor in DENV infection
Platelet activation factor (PAF) is the trivial name of a phospholipid that has the chemical structure of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, characterized by an alkyl ether bond at the sn-1 position in the glycerol chain (Fig. 3) 32 . The hexadecyl (16:0) moiety as the linker at the sn-1 position provides greater biological activity to the PAF; however, chain length specificity is low, and this leads to the natural formation of a significant amount of 1-O-octadecyl species, i.e. with an octadecyl (18:0) moiety as the linker at the sn-1 position 33 . Given the variation in the length of the side chain of O-alkyl bound at the sn-1 position, as well as the variation of the 2-acetyl chain attached at the sn-2 position, a series of different PAFs may be formed at the same time 34,35 . The formation of PAFs occurs either by de novo synthesis, or by the lipid remodeling pathway 36 . The formation of PAFs by de novo synthesis is related to the maintenance of the physiological concentrations of this mediator when the organism is in homeostasis, i.e. constitutive PAF concentrations 37 . The synthesis of PAFs from the remodeling pathway, on the other hand, is regulated by extracellular stimuli, i.e. under inflammation or infection, and is responsible for the PAF pool that occurs under these conditions 38,39 . The synthesis of a pool of PAFs occurring under infection/inflammation is mediated by the activation of cytosolic PLA2 (cPLA2), which recruits and hydrolyzes phosphatidylcholines for the formation of lysoPAF 38,39 . cPLA2 is a member of a superfamily of phospholipases responsible for the recruitment of a series of lipids involved in inflammatory and immune response processes 10 . Therefore, with the activation of cPLA2 due to extracellular stimuli (phosphorylation and Ca 2 + ), PAFs synthesis is initiated by the remodeling pathway, with the formation of 1-O-alkyl-sn-glycer-3-phosphocholine (lyso-PAF) from the hydrolysis of 2-Acyl-1-alkyl-sn-glycero-3-phosphocholine by cPLA2, which has the characteristic of hydrolyzing fatty acids linked to the sn-2 position of the glycerol chain 37 . Thus, the action of the platelet-activating factor acetylhydrolase, activated by Ca 2+ , and phosphorylation of lyso-PAF 40 , leads to PAF formation (Fig. 3).
PAFs exhibit biological activity in various cells and tissues 41 and the interaction of PAF agonists occur through the PAF receptor (PAFR). PAFR is comprised of seven transmembrane helixes coupled to the G protein, and is present in both plasma membrane and nuclear membrane. Activation of this receptor leads to the influx of Ca 2+ into the cell, promoting a series of simultaneous signaling of kinases and phospholipases, such as MAPK, PKC, phosphatidylinositol-3-kinase (PI3K), protein tyrosine kinase (PTK), phospholipase Cβ (PLC β), and PLA 42 . Thus, cPLA2 itself is activated by increasing Ca 2+ concentrations and phosphorylations promoted by the interaction of PAF with its receptor, generating a positive feedback for the production of a PAF pool 40,43 .
The simultaneous activation cascade resulting from PAF promotes a multitude of effects involved in the immune and inflammatory responses, such as the production of superoxide, thromboxane B2 and leukotriene C4 44 , increased endothelial permeability 45 , increased production of proinflammatory interleukins 46 , eosinophils recruitment 47 , among other effects involved in the immune response against viral infection. PAFR is present in the membranes of various cell types such as eosinophils, leukocytes, macrophages, neutrophils and platelets 43 . The correlation between PAF and platelet activation is important in the viral infection process, especially for DENV; SCIENtIFIC REPORTS | (2018) 8:11826 | DOI:10.1038/s41598-018-30385-x the endogenous release of PAF is related to a number of acute inflammation effects in DENV infection, such as increased vascular permeability, altered leukocyte numbers, thrombocytopenia, and degrees of bleeding 48 . Thus, the identification of 4 PAF precursors as biomarkers becomes a strong indication for representing this process.
It has recently been shown that platelets are also involved in the detection of pathogen-associated molecular patterns (PAMPs) 49 by standard-recognition receptors (PRRs) on the surface, such as Toll-like receptors (TLRs), and associated with other members of the interleukin-1 (IL-1)-TLR superfamily. Moreover, it has been reported that the number of constitutive PRR on the surface of the platelet undergoes specific upregulation when the platelet is active by a given microorganism 50 , which would increase the sensitivity to the immune response to the pathogen, assisting in the fight against infection. This mechanism indicates that platelets are part of the first-line recognition process for both microbial pathogens and immune response. Given the fact that platelets have direct antimicrobial functions mediated by the secretion of antimicrobial molecules, including platelet microbicidal proteins (PMPs) and kinocidins 49 ; Tsegaye et al. 2013 demonstrated that the release of CXCL4 by platelets inhibited in vitro HIV-1 infection in T cells. Thus, it is feasible to infer that platelet activation may play an important role during the viral immune response process 51 .
The identification of precursor species involved in the synthesis of PAFs in this study is in line with the findings proposed by Berthet et al. 2012, where peripheral blood platelets exposed to S. minnesota led to increased concentrations of IL-6, IL-8 and TNFα, whereas platelets exposed to E. coli did not 52 . This behavior evidences that the secretion of platelet cytokines is distinct due to the activation pattern, and suggests a specific response where lipids are responsible for such specificity 53,54 .
In contrast to helping fight viral infections, one of the major complications manifested as a result of the interaction between viral infection and platelets, and which is directly related to the activation of PAF, is thrombocytopenia 55,56 . This characteristic manifestation of DENV infection is even used as one of the criteria for the diagnosis of this infection 56 . Thrombocytopenia has been used as a parameter for the identification of patients with a more severe clinical picture, which also encompass other symptoms such as increased vascular permeability and hematocrit, alterations in the number of leukocytes and hemorrhage (varying degrees) 48 . A study by Yang et al. demonstrated greater release of PAF by macrophages in patients with DENV-1 virus than control subjects 57 . In addition, in studies with mice deficient in PAFR, primary infection by DENV was less severe. This occurs because inhibition of the PAF-PAFR interaction decreases the production of proinflammatory cytokines and TNF-α, in addition to decreasing vascular permeability 57,58 . The increase in intracellular Ca 2+ , which may be promoted by the PAF-PAFR interaction, is a crucial factor in the activation of platelet response, including the translocation of P-selectin to the membrane 59 .
Thrombocytopenia in DENV infection occurs due to the immunological destruction of virus-platelet complexes; viral activation of platelets induces overexpression of P-selectin, functioning as a receptor for macrophages 60 . In cases of recurrent DENV infections, antibodies against the prM structural viral protein facilitate efficient binding of their immature particles to cells expressing the Fc receptor, such as platelets (FccRIIa), which bind to these anti-prM-DENV complexes susceptible to destruction by the immune system 61 . In addition, the DENV-platelet complex also binds to complement C3 molecules and to platelet-associated IgM or IgG antibodies, resulting in their clearance by immune system cells 62 , which would result in thrombocytopenia. Additionally to the mechanisms of destruction of the platelet-DENV complex mentioned above, anti-DENV antibodies react against platelet glycoproteins mediating their destruction by the complement or monocyte-macrophage system 63 .
Therefore, all PAF precursors identified and elucidated in this study highlight the importance of increasing the synthesis of PAF performed by the remodeling pathway in the inflammatory process, confirming its activation by external factors 36 . In addition, the synthesis of PAF pools represents the extensive activation of PAFR, a factor related to the severity of dengue cases, which leads to increased cytokines, increased vascular permeability and, consequently, severe hemorrhage and shock 48 . Moreover, platelet activation correlates with the initiation and maintenance of the immune response, as platelets participate in the front line detection and initiation of the immune response 49 . Platelets still play an ambiguous role in the literature, in which they collaborate in the fight against the infection in the organism 49 , while at the same time are involved in the degree of severity of the disease, being responsible for provoking hemorrhagic fever due to thrombocytopenia 56 .
A previous contribution by our research group has confirmed that it is possible to carry out a study on viral infection mechanism through the direct analysis of the serum of infected patients 7 , assertively providing the metabolomics profile of the pathophysiology of the viral infection process, without further degrees of sample preparation and (pre-)processing. Remarkably, this is possible thanks to the integration between mass spectrometry and bioinformatics to analyze the large amount of data generated. All biomarkers were chosen and validated by statistical analysis and are in line with previous studies on the changes arising from DENV infection both in vivo and in vitro 19,20,25,64 . Thus, the use of these biomarkers opened the possibility to systemically assess the alterations on the lipid pool due to DENV infection, which occurred through the increase of PC synthesis and the recruitment of TGs to supply the bioenergetic needs due to the infection. This study also clarifies the possible mediators of the most severe form of the disease, the hemorrhagic form, since the direct analysis of serum allowed to identify a series of precursors of PAFs. According to data in the literature, the increase in PAFs is closely related to two of the main characteristics of this infection: hemorrhagic fever 16 and thrombocytopenia 59 . These symptoms, nonetheless, are not exclusively a result of DENV infection: they occur in other infectious diseases as well 55,56,61 . Hence, future efforts in identifying metabolites directly related to several infection processes, as performed herein, will allow us to verify whether the mechanism involved in these diseases is common or specific for each pathogen. Also, our results may enables and encourages the medical community to screen patients with conditions that have potential for hemorrhagic aggravations with a higher degree of confidence for clinical prognosis.  Table 2 organizes the structure of sample collection and provides a view of the total number of analyzed specimens. A written informed consent was obtained from all patients prior to enrollment. All samples were obtained from the Base Hospital of SJRP. All experiments were performed in accordance with relevant guidelines and regulations regarding samples from human origin.  Sample preparation. Serum preparation was performed as described by Melo et al. 2017 7 . In summary, 20 μL of each biological sample (blood serum) were diluted in 200 μL of tetrahydrofuran and homogenized and then the volume was completed to 1 mL with methanol, with further homogenization. The obtained solution was centrifuged and 20 μL of the supernatant was collected and diluted in 980 μL of methanol and 0.1% of formic acid was added to the final solution. Statistical analysis. Statistical analysis to choose chemical markers for each group was performed using Principal Component Analysis (PCA). PCA is a multivariate model of covariance structure modeling; it is used with the specific purpose of analyzing correlation structures, and it is characterized as a statistical analysis technique for potential biomarkers screening by a given "omic" platform 14 . To perform PCA analyses, raw data were used as a pool of all samples within the same data matrix, i.e. all mass spectrometric data from all replicates of both Control and DENV group were organized in a single database, which was inputted in the online platform environment. Prior to PCA analyses, interquartile range was used as data filtering method, with quantile normalization and auto scaling. A heatmap of the all features selected by PCA analyses was built using the Pearson's distance measurement and Ward's clustering algorithm. Fold Change analysis was performed for all features selected by PCA and elucidated by HRMS and MS/MS analysis. All statistical analyses were performed using the online platform MetaboAnalyst 3.0 66,67 .  (Table 1 and Supplementary Information 1).