Abstract

1. Introduction

Dengue virus (DENV) infection is one of the most rapidly growing and significant mosquito-borne infections in tropical and subtropical regions worldwide [1,2]. While primary DENV infection is typically self-limiting and may cause flu-like symptoms, secondary infections are associated with a higher risk of severe dengue, also known as dengue hemorrhagic fever (DHF). Currently, there are no specific antiviral treatments for DENV infection. Climate change, leading to variations in temperature and humidity, is likely expanding mosquito habitats and increasing the overall risk and mortality of DENV infections [3,4,5,6,7,8,9,10].

Severe dengue is characterized by endothelial damage and vascular leakage [11,12,13,14,15,16,17,18,19,20,21,22], which usually occur 3–6 days after the onset of disease during the acute phase of DHF [22,23]. The occurrence of this phase after the period of decreasing viremia suggests that the severe symptoms are due to endothelial injury rather than direct infection and replication of the DENV in the endothelium [22]. However, the specific viral factors causing vascular endothelial damage remain unclear.

The DENV envelope protein (E) domain III (EIII) is known for its role in host cell surface protein binding during viral entry [24]. Our previous in vitro studies showed that treatment with viral-load-equivalent levels of recombinant EIII (rEIII) can induce various regulated cell death (RCD) responses in endothelial cells, platelets, and neutrophils, indicating potential cytotoxicity of rEIII when it binds to these cells [25,26,27].

Given that secondary, but not primary, DENV infections are associated with DHF, and that abnormal immune responses are more intensely elicited during secondary infections, it is suggested that DHF is characterized not only by direct viral damage but also by these critical pathogenic immune responses [28,29,30,31,32,33]. DENV infection and immunization with viral protein nonstructural protein (NS1) can lead to the production of autoantibodies [e.g., anti-platelet immunoglobulins (Igs)], contributing to abnormal immune responses and excessive inflammation [29,31,34,35,36,37,38]. As vascular leakage follows peak viremia (EIII expressed on virion surfaces) and coincides with a marked increase in DENV-elicited antibodies [39], it is plausible that the sequential elevation of circulating EIII levels combined with DENV-elicited antibodies through injections results in endothelial damage and hemorrhage pathogenesis in vivo.

In previous studies, we demonstrated that sequential injections of DENV, rEIII, and anti-platelet Ig, mimicking the clinical phase of DHF with peak viremia followed by elevated DENV-induced Ig and autoantibodies [25,33,35], could induce hemorrhagic pathogenesis in mice. Intriguingly, this model replicates key DHF-associated responses, including vascular leakage, hemorrhage, thrombocytopenia, and excessive inflammation [25,33,35]. In this scenario, however, the specific form of RCD, such as apoptosis, contributing to endothelial cell death in vivo has not been clearly established. Therefore, our study aims to determine the role of apoptosis and explore whether it could be targeted for treating the combined effects of rEIII and autoantibodies in mice.

Experiments have used an increased expression of FcγRIII and tissue factor (TF), along with reduced thrombomodulin (TM) levels, as indicators of endothelial inflammation and damage [40,41,42,43], while active caspase-3 serves as a marker of endothelial apoptotic cell death [44,45]. In this study, to assess the cytotoxic effects of EIII on endothelial cells, we measured the expression levels of FcγRIII, TF, TM, and activated caspase-3 in cultured endothelial cells treated with rEIII and DENV virions. In the in vivo experiments, we examined whether sequential injections of rEIII and anti-platelet Ig in a mouse model could induce endothelial apoptosis associated with hemorrhage.

Our aim is to explore whether targeting endothelial cell death-inducing pathways could serve as a therapeutic approach to ameliorate EIII-induced endothelial injury and hemorrhage. To test this, we used inhibitors such as chondroitin sulfate B (CSB), which disrupts DENV-EIII binding [26,27], N-acetyl cysteine (NAC), an antioxidant that reduces oxidative stress during DENV infection [46], and a caspase-3 inhibitor z-DEVD that blocks apoptotic cell death [47,48,49,50]. This approach was based on our preliminary findings that rEIII induces endothelial apoptosis in vitro and that the combination of rEIII and autoantibodies causes endothelial apoptosis in mice, prompting the inclusion of a caspase-3 inhibitor in the study. Additionally, our previous research demonstrated that CSB, which blocks rEIII-endothelial interactions [27], and NAC, an antioxidant, suppressed rEIII-induced endothelial pyroptosis [25]. However, their ability to prevent rEIII-induced endothelial apoptosis was not yet clear. Thus, we used these agents to explore their effects on rEIII- and rEIII plus autoantibody-induced endothelial abnormalities in both the in vitro and in vivo models, with promising results in reducing endothelial damage. The regulatory mechanisms and therapeutic potential are discussed further.

2. Results

2.1. Treatments of Recombinant EIII Protein and Virion of DENV Markedly Induced Elevation of Endothelial Inflammation and Damage Markers in HMEC-1 Endothelial Cells

Employing a reductionist approach, we compared the effects of the rEIII and DENV virion treatments on human endothelial HMEC-1 cells to determine if rEIII is a key viral cytotoxic factor in DENV-induced endothelial pathogenesis. We assessed the surface expression of the endothelial markers FcγRIII, TF, and TM, which indicate inflammation and damage. Both rEIII and DENV virion treatments resulted in increased FcγRIII and TF expression, along with decreased TM levels (Figure 1; examples of flow cytometry gating for TM expression can be found in Figure S1). This aligns with previous studies showing that elevated FcγRIII and TF expression and reduced TM serve as indicators of endothelial inflammation and damage, respectively [40,41,42,43]. These results suggest that rEIII, a DENV-associated cytotoxic factor, can effectively induce endothelial inflammation and damage.

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Figure 1

Assessment of endothelial cell inflammation and damage markers. Cell populations with relatively high surface expression levels of endothelial cell inflammation and damage markers, FcγRIII, tissue factor (TF), and thrombomodulin (TM), of HMEC-1 cells were measured using flow cytometry following treatments with vehicle (solvent control), recombinant glutathione S-transferase (rGST), rEIII, and DENV. Vehicle control groups were set to a 1-fold baseline for normalization. Statistical significance is indicated by ** for p < 0.01 and * for p < 0.05, compared to untreated vehicle groups, and # for p < 0.05, compared to rGST groups. This study consisted of three experiments with two replicates per group (n = 6).

2.2. Treatments of Recombinant EIII Protein and Virion of DENV Markedly Induced Caspase-3-Associated Cell Death in HMEC-1 Endothelial Cells

We aimed to evaluate whether inhibitors of cell death pathways could serve as targeted therapeutic agents. However, the expression levels of FcγRIII, TF, and TM do not provide insights into cell death conditions (Figure 1). Consequently, we measured caspase-3 activity and cell survival in HMEC-1 cells following treatments with rEIII and DENV virus particles (Figure 2). We found that additional treatments with CSB, antioxidant NAC, interleukin (IL)-1 receptor antagonist (IL-1RA), tumor necrosis factor-α (TNF-α) inhibitor etanercept, and caspase-3 inhibitor z-DEVD-FMK (z-DEVD) markedly rescued cells from rEIII-, and DENV-induced caspase-3-associated death (Figure 2).

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Figure 2

Caspase-3-associated cell death occurred after HMEC-1 human microvascular endothelial cells were challenged with recombinant EIII (rEIII) and DENV virions. The recombinant rEIII and DENV treatments were compared to untreated groups and control groups treated with the recombinant protein rGST. The rescue effects of chondroitin sulfate B (CSB), antioxidant N-acetyl cysteine (NAC), IL-1 receptor antagonist (IL-1RA), TNF-α inhibitor etanercept, and caspase-3 inhibitor z-DEVD-FMK (z-DEVD) were assessed by measuring the suppression of cellular caspase-3 activities (A) and the reduction in cell death (B), compared to untreated groups. The untreated vehicle groups were set to 100% for normalization. ** indicates a statistically significant exacerbated effect at p < 0.01 compared to untreated vehicle groups; ## indicates a statistically significant ameliorated effect at p < 0.01 compared to respective vehicle groups. This study involved three experiments with two replicates per group (n = 6).

2.3. Treatment with Recombinant EIII Protein and Anti-Platelet Ig in a Mouse Model Markedly Induced Caspase-3-Associated Damage in the Vascular Endothelial Cells of Hemorrhage Lesions

Although our experiments with rEIII and anti-platelet Ig treatments have demonstrated that these dual challenges could induce hemorrhagic pathogenesis in mice [25], it remains unclear whether these treatments induce apoptosis in vivo. Given that the rEIII and DENV virions elicited similar pathogenic responses in endothelial cells (Figure 1 and Figure 2), we used a reductionist approach to evaluate if rEIII can induce hemorrhage-associated apoptotic cell death in vascular endothelial cells in vivo. In our mouse model, we applied dual challenges with rEIII and anti-platelet Ig and compared the outcomes with those from previously reported methods involving DENV virion and anti-platelet Ig dual challenges [35]. We employed immunohistochemistry (IHC) fluorescent staining to detect the endothelial cell marker CD31 and activated caspase-3 at hemorrhage lesion sites (Figure 3).

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Figure 3

Immunohistochemistry analysis of endothelial damage in hemorrhage lesions of mouse skin samples using confocal microscopy. Mouse skin samples were collected after subcutaneously challenged with vehicle (A), rEIII (first hit alone) (B), rEIII + control Ig (C), rEIII + anti-CD41 Ig (dual challenge) (D–F), and rEIII + anti-NS1 Ig (dual challenge) (G–I). Skin samples were then subjected to immunohistochemistry (IHC) (A–I) and quantitative (J–L) analyses. CD31, activated caspase-3 (a-caspase-3), and DAPI staining signals in the IHC images indicate the localizations of endothelial cells, apoptotic cells, and cell nuclei in the mouse skin sections, respectively. The asterisks (*) in (A–C,G) (red channels) mark fluorescence signals from muscle layers underneath the skin. White arrows in (A–D,G) (CD31 staining) indicate the locations of blood vessels. Panels (F) and (I) are enlarged merged images shown in (E) and (H), respectively. Scale bars: (A–D,G) 50 μm; (E,F,H,I) 10 μm. Quantitative results were obtained by Image J-abstracted signal intensities from respective channel images: activated caspase-3 (green channel)/CD31 (red channel) (J), activated caspase-3 (green channel)/DAPI (blue channel) (K), and activated caspase-3 (green channel)/nucleus counts (accountable nucleus number in the blue channel) (L), from five different 200× images (n = 5, in 2 independent experiments). Due to the high fluorescence background, muscle areas were excluded from quantitative analyses. Vehicle groups were normalized to 1-fold (J–L). ## p < 0.01 vs. vehicle groups.

We found that rEIII plus anti-platelet Igs (either anti-CD41 monoclonal Ig MWReg30 [51], or anti-DENV-nonstructural protein 1 Ig (anti-NS1 Ig), which contains anti-platelet Ig fractions [31,35]) induced a clear appearance and co-localization of CD31 and activated caspase-3 signals in the IHC fluorescent staining (Figure 3A–I, rEIII + control Ig groups vs. rEIII + anti-CD41 Ig, and rEIII + anti-NS1 Ig groups), with markedly elevated quantitative results (Figure 3J–L). These results suggest that rEIII plus the anti-platelet Ig challenges effectively induce caspase-3-associated apoptotic cell death in endothelial cells in vivo.

2.4. Treatment with Caspase-3 Inhibitor z-DEVD Markedly Mitigated Hemorrhage-Associated Pathogenesis Induced by the Recombinant EIII Protein and Anti-Platelet Ig Challenges in Mice

The caspase-3 inhibitor z-DEVD was used to determine whether caspase-3-dependent apoptosis contributed to the hemorrhage pathogenesis induced in vivo by the rEIII and autoantibody challenges, in line with our previously established mouse model [25]. While applying human guidelines to assess disease severity in mice is challenging, the pathophysiological alterations observed in this model bear some resemblance to those seen in DHF patients [35]. Following the dual challenges with rEIII plus either an anti-platelet monoclonal antibody or anti-dengue nonstructural protein 1 immunoglobulin (anti-NS1 Ig, which includes anti-platelet autoantibody fractions), the z-DEVD treatment markedly ameliorated all measured pathological manifestations in the mice (Figure 4). These included thrombocytopenia (Figure 4A outline, Figure 4B), hemorrhage (Figure 4C,D), inflammation (Figure 4E), hypercoagulation (Figure 4F), and endothelial damage (Figure 4G)—symptoms linked to DHF [52,53,54]. This implies that the caspase-3 pathway is involved in the disease mechanism, with caspase-3-associated cell death contributing to the pathogenesis driven by the rEIII and autoantibody challenges.

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Figure 4

Involvement in the caspase-3 pathway in pathogenesis induced by rEIII and autoantibody treatments is illustrated. The experimental outline (A) and various parameters, including mouse platelet counts (B), hemorrhagic lesion severity (C,D), proinflammatory cytokines TNF-α, IL-1β, and IL-6 (E), anticoagulants protein C and antithrombin III (F), and soluble thrombomodulin (G), are shown. Treatment with the caspase-3 inhibitor z-DEVD (B–G) reduced hemorrhage and inflammation in wild-type mice challenged with rEIII (the first hit) and autoantibodies [the second hit: anti-CD41 Ig (α-CD41) and anti-NS1 Ig (α-NS1), containing anti-platelet fractions]. This study used six mice per group, with three experiments conducted (n = 6). Data are presented as mean ± SE, with # p < 0.05 and ## p < 0.01 indicating significant rescue compared to the respective rEIII + α-CD41/α-NS1 groups. Ctrl Ig: control Ig. For detailed hemorrhage scoring, refer to Figure S2. The mouse diagram was created using Biorender.com.

2.5. Treatment with CSB and NAC Markedly Rescued Hemorrhage-Associated Pathogenesis Induced by Recombinant EIII Protein and Anti-Platelet Ig Treatments in Mice

As the treatment with CSB and NAC appeared to potentially rescue caspase-3-associated endothelial cell death in vitro (Figure 2), we aim to explore whether CSB and NAC treatments can also mitigate hemorrhage pathogenesis in vivo. Our findings revealed that treatments with CSB and NAC markedly ameliorated various pathological changes, including thrombocytopenia (outlined in Figure 5A, shown in Figure 5B), hemorrhage (Figure 5C,D), inflammation (Figure 5E), hypercoagulation (Figure 5F), and endothelial damage (Figure 5G), which are potential clinical indicators associated with DHF [52,53,54], induced by the dual challenge protocol.

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Figure 5

Rescue of pathogenesis induced by rEIII plus autoantibody dual challenges by CSB and NAC. The experimental layout (A) and the pathophysiological alterations in mouse platelet counts (B), hemorrhagic lesions (C,D), proinflammatory cytokines TNF-α, IL-1β, and IL-6 (E), anticoagulants protein C and antithrombin III (F), and soluble thrombomodulin (G) are depicted. (B–G) Treatments with chondroitin sulfate B (CSB), a glycosaminoglycan that binds to rEIII (0.5 mg/kg), and N-acetyl cysteine (NAC), a scavenger of reactive oxygen species (ROS) (50 mg/kg), were employed to alleviate hemorrhage and inflammation in wild-type (WT) mice subjected to the “rEIII (the first hit) plus autoantibody (the second hit)” dual challenge (panels 3 and 4, 6 and 7 vs. 2 and 5, respectively). The data are presented as mean ± SE. # p < 0.05 and ## p < 0.01 indicate significant rescue compared to the respective “rEIII + anti-CD41 Ig” or “rEIII + NS1 Ig” groups. Ctrl Ig: control Ig. This study included six mice per group, with a total of three experiments conducted (n = 6). The mouse diagram was created using Biorender.com.

In summary, our injection protocol in the mouse model, where rEIII is administered and initially followed by anti-platelet Ig 24 h later, somewhat reflects the clinical progression of DHF, in which viremia precedes the induction of DENV-associated autoantibodies and hemorrhage pathogenesis develops when both viremia and autoantibodies are present. Given that EIII-induced caspase-3 endothelial apoptosis is likely implicated in the endothelial cell damage (Figure 2), treatments with inhibitors like CSB, which target EIII-cell-surface binding, as well as the ROS scavenger NAC, and the caspase-3 inhibitor z-DEVD, which targets endothelial cell death (Figure 2, Figure 4 and Figure 5), may offer promising avenues for developing therapeutic strategies to mitigate hemorrhage pathogenesis induced by the dual challenges.

3. Discussion

In this study, we have demonstrated that recombinant DENV EIII, in conjunction with the autoantibody dual challenges, can induce caspase-3-dependent apoptosis in endothelial cells both in vitro and in vivo. This finding provides new insights into the molecular interactions between the DENV and host cell responses, potentially elucidating the mechanisms behind the vascular complications observed in severe DENV infections.

Dengue viruses typically do not induce DHF in wild-type mice through natural propagation [55,56]. However, following the established protocols, we can induce hemorrhagic pathogenesis by concurrently administering DHF-viral-load DENV and anti-platelet autoantibodies, leading to severe hemorrhage and a surge in cytokines [25,35]. In this study, we applied a reductionist approach and found that treatment with rEIII alone effectively substitutes DENV in inducing endothelial damage, as demonstrated in both the in vitro endothelial cell culture model (Figure 1 and Figure 2) and the dual challenge mouse model (Figure 3, Figure 4 and Figure 5), in comparison to DENV plus the autoantibody treatments from a previous report [35].

To better mimic the pathological changes in mice that resemble human responses, we employed a DHF-viral-load equivalent EIII dosage, following previously described methods [27]. This approach involved comparing the functionally equivalent levels of DENV and rEIII using an activated partial thromboplastin time (aPTT) plasma clotting assay, as rEIII can inhibit the anticoagulant effect of heparin [27]. To explore the dose-dependent response of DENV and rEIII on plasma clotting in heparinized mouse plasma, regression curves were plotted, comparing the DENV and rEIII dosages with the aPTT results. We found that both the DENV and rEIII doses were inversely correlated with the aPTT in a similar manner. Through this method, we determined the functionally equivalent doses of DENV and rEIII in their interaction with heparin. For instance, the estimated DHF viral load virion titer (1 × 10³ to 1 × 10⁶ PFU) [57] roughly corresponds to 1.7 × 10⁻³ to 1.2 μM rEIII. The 0.3–1.2 μM rEIII dose used for the analysis in this study falls within this range.

Our findings revealed that rEIII alone is sufficient to induce comparable levels of caspase-3 activation and apoptotic cell death in vitro, similar to the DENV virion treatments, indicating that virus replication is not required to cause endothelial cell damage. Notably, our in vivo protocol—administering rEIII followed by anti-platelet Ig 24 h later—in part mirrors the clinical progression of DHF, where viremia precedes DENV antibody production [39]. This sequence effectively induces caspase-3-associated endothelial cell damage (Figure 3) and hemorrhagic pathogenesis in mice (Figure 4 and Figure 5), resembling the traditional LPS-elicited Shwartzman reaction [35,58,59,60], which triggers TNF-α, IL-1, pro-coagulation activity, and hemorrhaging (Figure 4 and Figure 5). These results suggest that dengue-associated hemorrhage may stem from an rEIII and autoantibody-induced Shwartzman reaction-like response. Notably, compared to monoclonal anti-CD41 Ig, anti-NS1 Ig exhibited relatively lower potency in hemorrhage-related pathogenesis, such as the hemorrhage score, proinflammatory cytokine induction, and thrombomodulin release (Figure 4 and Figure 5). This may be attributed to the fact that, unlike monoclonal anti-CD41 Ig, only specific fractions of anti-NS1 Ig demonstrate anti-platelet activity, resulting in a less pronounced pathogenic effect in mice.

Endothelial dysfunction and vascular leakage are key characteristics of severe dengue disease, including DHF and dengue shock syndrome [23,61]. Previous studies have shown that elevated FcγRIII and TF expression, along with reduced TM levels, are markers of endothelial inflammation and damage [40,41,42,43]. Consistent with these observations, our findings demonstrate that the rEIII and DENV virion treatments lead to an increased expression of FcγRIII and TF and decreased TM expression, which are linked to endothelial apoptosis in vitro.

Endothelial apoptosis induced by DENV and DENV-elicited autoantibodies has been reported in various in vitro models [46,62,63,64,65,66,67,68], while the specific form of RCD, such as apoptosis, contributing to endothelial cell death in vivo remains unclear. In this study, we demonstrate for the first time that rEIII induces endothelial apoptosis in an rEIII plus autoantibody dual challenge mouse model. The marked rescue effect of the caspase-3 inhibitor z-DEVD, which prevents caspase-3 activation, suggests that apoptosis plays a critical role in initiating hemorrhagic pathogenesis. Despite DENV’s low replication levels in endothelial cells, indicating that endothelial damage is not solely due to viral replication, our findings show that rEIII alone can induce endothelial cell death, further supporting the idea that DENV replication is unnecessary for this damage. The caspase-3-mediated cell death observed in the cultured HMEC-1 cells, which was mitigated by the caspase-3 inhibitors, indicates that rEIII triggers the apoptotic pathway. Additionally, rEIII plus the autoantibody dual challenges induced apoptotic abnormalities in the CD31⁺ vascular endothelial cells at hemorrhagic lesions in the mouse model. Previous studies have demonstrated that CSB can inhibit EIII binding to host cells [25], which explains the rescue effects of CSB observed in both the in vitro and in vivo experiments (Figure 2 and Figure 5).

In this present study, we found that NAC, IL-1RA, and etanercept, which suppress ROS and the proinflammatory cytokines IL-1 and TNF-α, respectively, were able to rescue rEIII-induced caspase-3-associated cell death (Figure 2). This suggests that rEIII-induced caspase-3 activation may be mediated through ROS and inflammation, leading to cellular disruption and apoptosis. While the beneficial effects of IL-1RA and etanercept were previously demonstrated [25], our current findings further highlight NAC as a potent agent in mitigating rEIII plus autoantibody-induced hemorrhagic pathogenesis in mice (Figure 5). Consistent with prior research, IL-1RA and etanercept enhance endothelial cell survival by blocking IL-1 and TNF-α, and NAC’s protective effect is plausible given the link between excessive ROS levels and apoptosis [69,70]. Additionally, the protective role of CSB, which blocks EIII’s interaction with target cells [26,27], is consistent with its ability to safeguard rEIII-treated endothelial cells. Further studies are needed to elucidate the precise molecular events following the rEIII–autoantibody interaction.

Given the role of rEIII and autoantibodies in inducing apoptosis, interventions that can block this interaction may serve as potential therapeutic strategies. Developing inhibitors to prevent rEIII and autoantibody-induced pathogenic effects could mitigate endothelial damage and improve patient outcomes. Our findings demonstrate that inhibiting rEIII binding with CSB, suppressing ROS with NAC, and blocking caspase-3 apoptosis with z-DEVD markedly rescue hemorrhagic pathogenesis in the mouse model. These results suggest that targeting EIII, ROS, and caspase-3 apoptosis may be promising approaches for developing drugs to treat DENV-induced hemorrhagic pathogenesis. Additionally, CSB, NAC, and z-DEVD could serve as promising reference compounds for the further development of therapeutic agents to control DHF.

Here, we demonstrate that the rEIII and autoantibody dual challenges successfully induce hemorrhagic pathogenesis in mice, which can be mitigated using various drugs and inhibitors. This model shows promise for studying the mechanisms of dual-challenge-induced pathogenesis and potential rescuing agents. However, since mice are not the native hosts of DENV and DENV cannot typically replicate in them, using cultured cells and mouse models may not fully capture the complex human immune response to DENV infection and DHF pathogenesis. Future research is needed to validate these findings in human subjects and explore the clinical relevance of EIII and autoantibodies in DHF pathogenesis.

4. Materials and Methods

Acknowledgments

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms251910858/s1.

Funding Statement

This work was supported by research funding from the National Science and Technology Council, Taiwan (104-2320-B-320 -009 -MY3, 107-2311-B-320-002-MY3, 111-2320-B320-006-MY3, and 112-2320-B-320-007), and the Tzu-Chi Medical Foundation (TCMMP104-06, TCMMP108-04, TCMMP 111-01, TCAS111-02, TCAS-112-02, TCAS113-04, TCRD112-033, and TCRD113-041). The funders had no role in the study design, in the collection, analyses, or interpretation of data, in writing the report, or in the decision to submit the article for publication.

Author Contributions

Conceptualization, project administration, and manuscript writing: H.-H.C.; formal analysis and investigation: T.-S.L., D.-S.S. and W.-S.W. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The Animal Care and Use Committee of Tzu-Chi University, Hualien, Taiwan, approved all the experimental procedures for investigating these animals, with an approval ID of 110081.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Footnotes

References