Editor's evaluation: Targeting the Annexin A1-FPR2/ALX pathway for host-directed therapy in dengue disease
Year: 2021
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Host immune responses contribute to dengue’s pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, interferon (alpha and beta) receptor one knockout (KO), AnxA1 KO, and formyl peptide receptor 2 (FPR2) KO mice were infected with dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac2-26 for analysis. In addition, the effect of Ac2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo. We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. Whilst the absence of AnxA1 or its receptor FPR2 aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestationsatteanuated the symptoms of the disease. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease. Editor's evaluation Costa, Sugimoto and colleagues report that the levels of anti-inflammatory Annexin A1 are reduced in dengue patients and in mice infected with dengue virus. They further show that absence of Annexin A1 or its FPR2/ALX receptor is associated with more severe disease, while treatment with an agonistic peptide had beneficial effects in mouse models. Their results suggest that strengthening Annexin A1 function may help to prevent severe dengue disease and agree with previous findings showing that treatment with corticosteroids that induce Annexin A1 may prevent the progression to severe illness. https://doi.org/10.7554/eLife.73853.sa0 Decision letter eLife's review process Introduction Dengue is caused by one of four serotypes of dengue virus (DENV1-4) transmitted by Aedes Aegypti and A. Albopictus mosquitoes, affecting around 400 million people in 128 countries (Bhatt et al., 2013). Occasionally, dengue infection develops into a potentially lethal complication identified as severe dengue, typified by exacerbated systemic inflammation, vascular leakage, fluid accumulation, respiratory distress, severe bleeding, and/or organ impairment (WHO, 2009). No antiviral drug for dengue treatment is available, and the use of Dengvaxia (CYD-TDV), the first dengue vaccine approved by the US Food and Drug Administration, has its limitations, such as the increased risk for development of severe dengue in the immune populations (Huang et al., 2021; WHO, 2018; The Lancet Infectious Diseases, 2018). Thus, the combination of these factors points to dengue as a major unmet clinical problem in countries affected by this disease (Shepard et al., 2016; Stanaway et al., 2016). The pathogenesis of severe dengue results from exacerbated host innate and adaptative immune responses to DENV. Among the target cells for DENV in humans, mast cells (MCs) lining blood vessels undergo dramatic cellular activation in response to DENV, despite their high resistance to infection (; St John et al., 2013bSt John et al., 2011). As essential regulators of vascular integrity, MC activation evoked by DENV triggers the production of inflammatory cytokines (cytokine storm) and vascular leakage, which ultimately result in hypovolemic shock in severe dengue (St John et al., 2013b; Syenina et al., 2015St John et al., 2011;). This is substantiated by a positive correlation between circulating chymase levels and disease severity recently observed in paediatric and adult patients (Rathore et al., 2020; Tissera et al., 2017). In line with the suggested role of uncontrolled immune responses in the pathogenesis of DENV infection, pharmacological suppression of inflammation (Fu et al., 2014; Marques et al., 2015; Souza et al., 2009), MC stabilisation (St John et al., 2013bMorrison et al., 2017), and inhibition of MC-derived protease (Rathore et al., 2019) have shown to be beneficial in experimental dengue. These studies provide proof-of-concept that host-directed therapies targeting excessive or misplaced inflammation may be a viable approach in treating severe dengue infection. Pro-resolving mediators are cell signalling molecules synthesised in a strict temporal and spatial fashion to regulate the host response and prevent the excessive acute inflammatory reaction that damages the host (Sugimoto et al., 2019). The discovery of this active phase of inflammation has led to a new awareness of how a disease can emerge, including the concept that dysregulation or ‘failure’ in pro-resolving mechanisms might be involved in the pathogenesis of several chronic inflammatory disorders (Eke Gungor et al., 2014; Fredman et al., 2016; Murri et al., 2013; Schett and Neurath, 2018; Sena et al., 2013; Thul et al., 2017; Vong et al., 2012). The pro-resolving protein Annexin A1 (AnxA1) and its cognate receptor formyl peptide receptor 2 (FPR2), also known as FPR2/lipoxin A4 receptor (FPR2/ALX), bear anti-inflammatory properties in sterile settings (Fredman et al., 2015; Galvão et al., 2017; Gimenes et al., 2015; Kusters et al., 2015; Leoni et al., 2015; Locatelli et al., 2014), and exert a degree of protection in infectious settings, such as experimental tuberculosis (Tzelepis et al., 2015; Vanessa et al., 2015), sepsis (Damazo et al., 2005; Gobbetti et al., 2014) Spite et al., 2009, pneumococcal pneumonia (Machado et al., 2020; Tavares et al., 2016), and influenza (Schloer et al., 2019). AnxA1 has been recently described to act as an endogenous modulator of MC degranulation in response to IgE/anti-IgE or compound 48/80 (Parisi et al., 2019; Sinniah et al., 2019; Sinniah et al., 2016; Yazid et al., 2013). Since AnxA1 is well known to counter regulate overexuberant pro-inflammatory events and MC activation, we have hypothesised that an imbalance between this anti-inflammatory/pro-resolving mediator and pro-inflammatory molecules could be operating during dengue infection. Whilst studies on bacterial infection consistently revealed the ability of specialised pro-resolving mediators and AnxA1 (Chiang et al., 2012; Decker et al., 2021; Gobbetti et al., 2014; Machado et al., 2020; Sekheri et al., 2020; Tzelepis et al., 2015; Vanessa et al., 2015) to facilitate innate immune responses against the pathogen whilst reducing the harmful effects of inflammation, the translational potential of these results to viral infection was less clear. In the present work, we have analysed the role of the pro-resolving AnxA1-FPR2/ALX pathway as a regulator of excessive inflammation observed in patients with the most severe forms of dengue infection. Our results suggest that failure to trigger this molecular pathway may contribute to disease severity in dengue infection and support the AnxA1-FPR2/ALX pathway as a potential target for host-directed therapy in human dengue disease. Results AnxA1 plasma levels are reduced in dengue patients To ascertain how DENV infection impacts the expression dynamic of the pro-resolving molecule, AnxA1, in humans, we measured the AnxA1 protein level in the plasma of DENV-infected patients (Figure 1A). Dengue patients (n = 60) were grouped into non-severe dengue (SD) (dengue patients that were treated at home as outpatients, n = 31) and SD (dengue inpatient cases that met WHO criteria for hospitalisation, n = 29) (Gonçalves Pereira et al., 2020). Demographics and laboratory characteristics are available in Table 1. Groups were comparable for sex distribution. In line with the association between older age in adults and an increased risk factor for progression to severe disease, our cohort was characterised by increased age in patients with SD (Rowe et al., 2014; Sangkaew et al., 2021). The proportion of patients showing secondary dengue infection was not significantly different between the non-SD and SD groups. All patients with positive polymerase chain reaction (PCR) reactions were infected with DENV-1, in line with a report that DENV-1 was the predominant serotype circulating in the city in which patients were recruited in the years of sample collection (Gonçalves Pereira et al., 2020). Outpatient evolution was confirmed by remote monitoring at the convalescent phase. Interestingly, we have identified that plasma levels of AnxA1 were reduced in dengue patients compared to healthy controls (Figure 1A). Stratification of groups according to disease severity at discharge showed that SD patients had discrete but significantly lower levels of AnxA1 compared to individuals with classic dengue (Figure 1A). Figure 1 Download asset Open asset Annexin A1 (AnxA1) levels are reduced in dengue patients and mice infected with dengue virus (DENV). (A) AnxA1 plasma levels in healthy and dengue patients. The latter group was stratified into non-severe dengue (non-SD, outpatients) and severe dengue (SD, inpatients) individuals. Each circle represents an individual participant and horizontal bars represent mean values for AnxA1 (ng/mL of plasma), assayed by ELISA. ****p<0.0001, **p<0.01 by two-tailed Mann–Whitney test. (B) Five-week-old BALB/c (n = 8) or (C) C57BL/6 (n = 5–6) WT mice were intravenously injected with 1 × 106 PFU of DENV-2 and culled in the indicated time points for plasma collection. AnxA1 plasma levels analysed by ELISA are shown. ***p<0.001 and ****p<0.0001 versus mock-infected group; #p<0.05, ##p<0.01 and ####p<0.0001 versus 24-hr-infected group; &&p<0.01 and &&&&p<0.0001 versus 48-hr-infected group; $$$p<0.001 versus 72-hr-infected group (one-way ANOVA followed by Tukey’s post hoc test). Figure 1—source data 1 Raw data for Figure 1A–C. https://cdn.elifesciences.org/articles/73853/elife-73853-fig1-data1-v2.xlsx Download elife-73853-fig1-data1-v2.xlsx Table 1 Demographics and laboratory characteristics of the study population from the control group, non-severe dengue (non-SD, outpatients), and severe dengue (SD, inpatients) groups during seasonal transmission 2013–2016. Characteristics and diagnosis of the study populationControl (n = 41)Non-SD (n = 31)SD (n = 29)Age a30 (19–58) **31 (17–65) *42 (19–76)Gender (F)71% (29/41)55% (17/31)52% (15/29)RT-PCR (n)0% (0/41)74% (23/31)38% (11/29)ELISA IgM (n)0% (0/41)61% (19/31)100% (29/29)Blood collection 1–5 days after symptom onset-58% (18/31)45% (13/29)Blood collection 6–12 days after symptom onset-42% (13/31)55% (16/29) A geometric mean (min-max). Blood collection: Fisher Test p>0.4. Age: Kruskal–Wallis Test p<0.003; Gender: Fisher Test p>0.1. DENV infection reduces AnxA1 plasma levels in mice Since we identified reduced levels of AnxA1 in the plasma of dengue patients, we sought to investigate the role of this pro-resolving mediator in dengue’s pathogenesis. To examine how the AnxA1 pro-resolving pathway operates in an immunologically intact system, we initially examined AnxA1 secretion over time in an immunocompetent animal model of DENV infection. Although wild-type (WT) mice are more resistant to infection than immunocompromised animals (Shresta et al., 2004); these animals were proven to be productively infected by DENV and are valid hosts to investigate the mechanisms underlying DENV-induced vascular dysfunction (Chen et al., 2007; St John et al., 2013b; Syenina et al., 2015). In both DENV-infected BALB/c (Figure 1B) and C57BL/6 mice (Figure 1C), there was a time-dependent decline in the concentration of plasma AnxA1 compared to mock-infected animals. Curiously, AnxA1 levels in human plasma were higher than in animal samples. Differences in AnxA1 plasma levels between both species have been previously noted (Senchenkova et al., 2019; Xu et al., 2021). The decline in AnxA1 expression during DENV infection motivated investigation on the role of the AnxA1-FPR2/ALX pathway in dengue disease progression and severity. Disruption of the AnxA1-FPR2/ALX pathway aggravated illness and exacerbated inflammation in DENV-infected mice We then conducted experiments in different animal strains to ascertain whether changes in AnxA1 expression could impact the dynamics of DENV infection (Figure 2A). As previously described (St John et al., 2013b), systemic infection of WT mice caused haematological and vascular changes consistent with the human disease, systemic MC activation, and inflammatory response (Figure 2B–K). Disease parameters were aggravated in AnxA1 KO animals compared with WT mice. In the absence of AnxA1, animals showed more severe and prolonged thrombocytopenia, haemoconcentration, and vascular permeability than WT mice (Figure 2B–D), indicating a protective role of AnxA1 in dengue disease. These findings were similar in FPR2-depleted animals (Figure 2G–I), suggesting that these effects are due to disruption of the AnxA1-FPR2/ALX pathway. Plasma levels of mast cell protease 1 (MCPT-1) and CCL2 were increased in either AnxA1 and FPR2 KO animals compared to their respective controls after infection (Figure 2E, F, J and K). While parameters, such as haematocrit and vascular leakage, returned to basal levels 72 hr after infection in WT animals, KO animals persisted with elevated levels, indicating a delay in resolving the host’s haematological and immune response. Interestingly, AnxA1-depleted animals have preserved their ability to control virus spread, as splenic virus replication was similar to what was found in control animals (Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset Mice are more susceptible to dengue virus (DENV-2) infection in the absence of Annexin A1 (AnxA1) or its receptor formyl peptide receptor 2 (FPR2). (A) Experimental design. (B–F) Five-week-old BALB/c WT and AnxA1 KO or (G–K) C57BL/6 and FPR2 KO mice were intravenously inoculated with 1 × 106 PFU DENV-2. Mice were culled in the indicated time points after infection and blood and tissue were collected for the following analysis: (B,G) platelet counts, shown as the number of platelets × 103/μL of blood; (C,H) haematocrit levels, shown as % volume occupied by red blood cells; (D,I) vascular leakage assay with Evans blue dye; concentrations of (E,J) MCPT-1 and (F,K) CCL2 in plasma, quantified by ELISA and shown as quantity per mL of plasma. B–C, n = 6–8 animals per group; D–K, n = 5. Differences over time were compared by two-way ANOVA followed by Turkey’s multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 versus mock-infected group; #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 versus 24-hr-infected group; &p<0.05, &&p<0.01, &&&p<0.001, and &&&&p<0.0001 versus 48-hr-infected group; $p<0.05, $$p<0.01, $$$p<0.001, and $$$$p<0.0001 versus 72-hr-infected group. Differences between genotypes were compared by two-way ANOVA followed by Sidak’s multiple comparison test, as indicated in the graphs. . Figure 2—source data 1 Raw data for Figure 2B–K. https://cdn.elifesciences.org/articles/73853/elife-73853-fig2-data1-v2.xlsx Download elife-73853-fig2-data1-v2.xlsx AnxA1 agonism attenuates DENV infection manifestations Given the modulation of AnxA1 expression in DENV-infected mice and the protective role of the AnxA1-FRP2/ALX pathway in experimental disease, we next questioned whether exogenous administration of the AnxA1 mimetic peptide Ac2-26 could attenuate dengue disease. Ac2-26 binds to FPR2/ALX and mimics most of the whole protein’s effects in experimental inflammation (Sheikh and Solito, 2018; Sugimoto et al., 2016). The peptide therapy in WT animals (Figure 3A) was effective after the first injection and protected against the major changes in haematological and immune markers as evident at 24 hr and 48 hr after DENV infection compared to mock-infected animals (Figure 3B–F). Thrombocytopenia (Figure 3B) and haemoconcentration (Figure 3C) were significantly milder in the group treated with Ac2-26. DENV-2 infection provoked increased vascular permeability in both untreated and treated animals, but to a lesser extent when animals were administrated daily with AnxA1 mimetic peptide (Figure 3D). Of note, whilst increased haematocrit levels and vascular leakage were observed as early as 24-hr post-infection in vehicle-treated mice, treatment with AnxA1 mimetic peptide delayed the onset of both disease manifestations (Figure 3B–C). Treatment with Ac2-26 also reduced the elevation in plasma MCPT-1 (Figure 3E) and CCL2 (Figure 3F) levels compared to the untreated group. Notably, the improvements in disease symptoms appeared to be independent of the splenic viral load (Figure 3—figure supplement 1). Figure 3 with 2 supplements see all Download asset Open asset Annexin A1 mimetic peptide Ac2-26 improves DENV-induced manifestations in wild-type (WT) mice and does not affect animals lacking its receptor formyl peptide receptor 2 (FPR2). (A) Experimental design. Five-week-old BALB/c WT (B–F) and FPR2 KO (G–K) mice were intravenously inoculated with 1 × 106 PFU DENV-2. Mice were treated or not with 150 μg Ac2-26 at the time of infection and daily thereafter by the intraperitoneal route. Mice were culled in the indicated time points after infection, and blood and tissue were collected for the following analysis: (B,G) platelet counts, shown as the number of platelets × 103 /μL of blood; (C,H) haematocrit levels, shown as % volume occupied by red blood cells; (D,I) vascular leakage assay with Evans blue dye; concentrations of (E,J) MCPT-1 and (F,K) CCL2 in plasma, quantified by ELISA and shown as quantity per mL of plasma. N = 5 animals per group, except for graph C where n = 4–5. Differences over time were compared by two-way ANOVA followed by Turkey’s multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 versus mock-infected group; #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 versus 24-hr-infected group; &p<0.05, &&p<0.01, &&&p<0.001, and &&&&p<0.0001 versus 48-hr-infected group; $p<0.05, $$p<0.01, $$$p<0.001, and $$$$p<0.0001 versus 72-hr-infected group. Differences between genotypes were compared by two-way ANOVA followed by Sidak’s multiple comparison test, as indicated in the graphs. Figure 3—source data 1 Raw data for Figure 3B–K. https://cdn.elifesciences.org/articles/73853/elife-73853-fig3-data1-v2.xlsx Download elife-73853-fig3-data1-v2.xlsx When we applied the same treatment schedule to FPR2 KO mice infected with DENV-2, Ac2-26 did not affect the disease parameters under observation (Figure 3G–K). Finally, the efficacy of Ac2-26 in these settings allowed us to validate the data obtained with AnxA1 KO mice, as administration of AnxA1 peptide to animals deficient in AnxA1 rescued the phenotype showed by this transgenic colony in dengue infection, bringing values of haematological and immune parameters in line with those measured in untreated mice (Figure 3—figure supplement 2). Taken together, these results reveal the therapeutic potential of a pro-resolving peptide in the context of dengue, supporting the hypothesis that it could be operative also in settings with lower or absent AnxA1. Protective effects of Ac2-26 are independent of the control of viral loads and virus dissemination To establish whether Ac2-26 treatment could have a therapeutic benefit after the infection is established and if it affects viral loads, we applied a different experimental system (Figure 4A), using mice bearing a null mutation for the interferon (alpha and beta) receptor 1 (IFNα/β KO; A129 mice). These animals are highly susceptible to DENV infection and present severe macroscopic and microscopic alterations (Costa et al., 2013; Costa et al., 2014; Lam et al., 2017; Shresta et al., 2004). As seen for BALB/c and C57BL/6 strains, DENV-infected A129 mice showed reduced AnxA1 plasma levels over the of infection, compared with mock-infected animals (Figure While untreated A129 mice of their from 2 post-infection the time treatment with Ac2-26 delayed onset (Figure The AnxA1 mimetic significantly reduced and haemoconcentration in response to DENV infection (Figure and and efficacy on innate with more effects in values than plasma levels for and (Figure and As observed in immunocompetent mice, Ac2-26 attenuated the systemic of MCPT-1 by DENV-2 (Figure it did not affect systemic levels of CCL2 (Figure Given the of early DENV infection in our it is that the blood level of these markers not be altered by the analysed time of A129 mice a degree of by elevated plasma levels of and (Figure treatment with Ac2-26 attenuated caused by DENV, as indicated by reduced (Figure and and levels (Figure Ac2-26 the absence of did not inflammatory and haematological parameters, or caused (Figure and In this animal and using this we could the effects of Ac2-26 following infection with DENV serotypes and observed a in haematological and production by either DENV-1, or (Figure Figure Download asset Open asset The protective effect of Ac2-26 administration in dengue virus A129 mice is viral (A) Experimental design. A129 mice were mock-infected or inoculated with 1 × 103 PFU of DENV-2 by the route. mice, were treated or not a with 150 μg of Ac2-26 by the intraperitoneal route. (B) Mice were culled in the indicated time points after infection, and plasma was collected for AnxA1 by ELISA (n = (C) was assessed in the indicated time points and as a of Ac2-26 red and mice treated with or Ac2-26 n = days after infection, animals were and blood and tissue collected for the following analysis: platelet counts, shown as the number of platelets × 103 /μL of blood (n = haematocrit levels, shown as % volume occupied by red blood cells (n = concentrations of and in plasma and of and DENV-infected mice, treated or not with Ac2-26. Plasma of MCPT-1 and in plasma of and DENV-infected mice, treated or not with Ac2-26. and were assessed by ELISA and are shown as quantity per mL of plasma or per of the n = n = n = n = of control and mice, treated or not with the AnxA1 were and into and of with and Plasma as n = loads from plasma, and of infected mice treated or not with examined by assay in Results are shown as the of of plasma or as the of of and (n = All results are as mean In over time and between were compared by ANOVA followed by Tukey’s multiple test: ****p<0.0001 versus mock-infected animals or the different as indicated in the In data were analysed by ANOVA followed by Tukey’s multiple test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 versus mock-infected group; #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001 versus mock-infected group treated with Ac2-26. In were by for Figure data 1 Raw data for Figure and Download Figure 5 Download asset Open asset Treatment with Ac2-26 disease by different dengue virus (DENV) significantly viral A129 mice were mock-infected or inoculated with × PFU of DENV-1, 1 × 103 PFU of or 1 × PFU of by the (n = mice were treated with or 150 μg of intraperitoneal a red days after infection, animals were and blood and tissue were collected for the following analysis: (A) counts, shown as the number of platelets × 103 /μL of (B) levels, shown as % volume occupied by red blood (C) Plasma as of in plasma and of and DENV-infected mice, treated or not with assessed by ELISA. are shown as of plasma or as of the loads from plasma and of mice infected with the serotypes of DENV and treated or not with by assay in Results are shown as the of of plasma or as the of of and In data were analysed by two-way ANOVA followed by **p<0.01, ***p<0.001, and ****p<0.0001 versus mock-infected or between infected mice treated with or as indicated in the multiple comparison test. In was by two-way ANOVA followed by multiple comparison test, and between animals treated with or Ac2-26 are indicated in the graphs. bars represent mean Figure data 1 Raw data for Figure Download Finally, we the potential impact of the Ac2-26 peptide on viral loads and virus A129 mice showed systemic viral on after with and viral load in and and Treatment with Ac2-26 did not affect systemic viral as untreated and treated mice showed similar and viral Treatment with Ac2-26 caused a in viral loads in the of mice infected with DENV-2 (Figure and in the plasma of animals inoculated with (Figure These data that the AnxA1 mimetic impacts this severe dengue or control on virus dissemination and viral loads, the host response. we show that the efficacy of the AnxA1 peptide is not to a virus Ac2-26 mast cell degranulation evoked by DENV is that mast cell (MC) degranulation to DENV-induced vascular leakage and disease severity (St John et al., 2013b; Syenina et al., 2015; Tissera et al., 2017). In line with we report increased MCPT-1 levels in the plasma of DENV-infected mice, which is in the absence of and is by AnxA1 peptide treatment (Figure and Figure J and Figure 3—figure supplement We hypothesised that the AnxA1 peptide at in exert its protective effects by MC degranulation. To this we of in and in vitro We first analysed from of WT mice with or AnxA1 mimetic peptide and infected with DENV (Figure Both animals treated or had MC degranulation in comparison with control mice. that systemic administration of Ac2-26 mast cell degranulation in we AnxA1 KO and FPR2 KO mice to the same and treatment schedule (Figure and We that Ac2-26 reduces mast cell degranulation in AnxA1 KO mice (Figure but to DENV-induced degranulation in FPR2 KO mice (Figure the of this receptor for its therapeutic Notably, the absence of FPR2 significantly increased mast cell degranulation in response to the with DENV compared to the WT (Figure Figure with 1 supplement see all Download asset Open asset Ac2-26 mast cell degranulation by dengue virus (DENV) both in and in (A) WT BALB/c mice were treated with Ac2-26 or
Source: eLife
Author: Frank Kirchhoff
Institution: Universität Ulm
Language: English
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4226159805