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.

We present evidence that an inadequate engagement of the resolution circuit AnxA1-FPR2/ALX may contribute to dengue infection’s pathogenesis with particular relevance for the cohort of patients affected by the most severe forms of the disease. By exploring resolution biology as a novel approach in dengue disease, both with respect to etiopathogenesis and pharmacological opportunity, we have identified that: (i) AnxA1 is downregulated in the plasma of dengue patients in comparison to healthy individuals and in DENV-infected mice in comparison to non-infected animals; (ii) depletion of the AnxA1-FPR2/ALX pathway aggravates clinical signs and enhances MC activation associated to DENV infection, indicating a nonredundant role for this resolution pathway in the pathogenesis of dengue disease; (iii) pharmacological treatment of mice with an FPR2/ALX agonistic peptide produced beneficial effects during DENV infection; (iv) AnxA1 mimetic peptide has direct inhibitory effects on MC degranulation induced by DENV while (v) it does not seem to control viral load and virus dissemination significantly.

In recent years, a new paradigm shift has emerged in our understanding of the pathogenesis of inflammatory diseases, which results from persistent and exacerbated pro-inflammatory signals and dysregulation or ‘failure’ in resolving mechanisms (Perucci et al., 2017) 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 severity and lethality of several infectious diseases, like dengue and the flu, frequently arise from an excessive host response, characterised by an uncontrolled release of pro-inflammatory cytokines leading to over-exuberant immune activation (Costa et al., 2013; St John et al., 2013a). Inflammation is physiologically balanced by resolution circuits, such as those centred on AnxA1 and lipid mediators (e.g. lipoxins, resolvins, protectins and maresins) that drive termination of inflammatory response yet helping the host to deal with the infective agent (Basil and Levy, 2016). In line with this, a growing body of evidence indicates that pro-resolving mediators are regulated during infection, contributing to the control and resolution of experimental and human infectious diseases (Abdulnour et al., 2016; Basil and Levy, 2016; Chiang et al., 2012; Frediani et al., 2014; Oliveira et al., 2017; Shirey et al., 2014). On the other hand, dysregulation in the production and/or action of pro-resolving mediators might contribute to the pathogenesis of sterile (Eke Gungor et al., 2014; Fredman et al., 2016; Murri et al., 2013; Sena et al., 2013; Thul et al., 2017; Vong et al., 2012) and infectious (Cilloniz et al., 2010; Colas et al., 2019; Molás et al., 2020; Morita et al., 2013) diseases, including atherosclerosis, inflammatory bowel diseases and tuberculous meningitis. Modulation of AnxA1 in the context of viral infections has been less investigated, and clinical data are scarce (Arora et al., 2016; Molás et al., 2020). Since recent evidence suggests a defective engagement of pro-resolving pathways during self-resolving infections (Cilloniz et al., 2010; Colas et al., 2019; Molás et al., 2020; Morita et al., 2013), we queried whether such alterations could also be occurring in dengue disease. Focus was given to the pro-resolving protein AnxA1 and its cognate receptor FPR2/ALX, a pathway that has been shown to exert a degree of protection in experimental tuberculosis (Tzelepis et al., 2015; Vanessa et al., 2015), sepsis (Damazo et al., 2005; Gobbetti et al., 2014), pneumococcal pneumonia (Machado et al., 2020; Tavares et al., 2016), and influenza (Schloer et al., 2019). Our data indicate that in addition to the already described early induction of an inflammatory response that may be harmful instead of protective, DENV infection is also characterised by sustained downregulation of molecular components of the AnxA1 pathway. The drop in circulating AnxA1 below basal levels observed in dengue patients in this study, especially in severe dengue, suggests that the pathogenesis and severity of the disease might be associated with a failure to engage mechanisms involved in endogenous anti-inflammatory signals and its resolution, such as those centred on AnxA1. Furthermore, we have identified a protective role for the AnxA1-FPR2/ALX pathway in DENV infection, as animals presented heightened signs of disease in the absence of either ligand or receptor, compared to WT mice. In line with integrated pro-resolving properties of resolution circuits, AnxA1 and FPR2 KO mice displayed diverse uncontrolled responses leading to increased disease severity. Markers of MC degranulation and systemic inflammation were elevated in transgenic mice compared to WT counterparts. Similarly, these genetically manipulated animals presented enhanced haematological alterations in our model of experimental dengue.

Infections are currently treated by drugs that target pathogens or inhibit their growth. In some infectious diseases, blocking inflammation pathways may be beneficial. While this approach might be successful in some infections, excessive inhibition of the immune response can also be associated with immunosuppression and increased mortality, as observed in septic patients (Carlet et al., 2020). Resolution pharmacology has been proposed as an alternative to balance the host response without hampering its ability to deal with the infection. Indeed, pro-resolving receptor agonists’ exogenous administration has proven benefits in experimental infectious settings, including bacterial pneumonia (Abdulnour et al., 2016; Machado et al., 2020) and influenza (Morita et al., 2013; Ramon et al., 2014; Schloer et al., 2019). In the present work, we provide proof-of-principle that a pro-resolving tool, the Ac_2-26 peptide, can ameliorate clinical disease and reduce circulating inflammatory mediators in an FPR2/ALX-dependent manner. The AnxA1 peptide was effective even when administered from day 2 after the infection onset.

In our experimental models, viral load was seemingly unaffected by Ac_2-26 treatment, supporting a protective effect independent of viral infectivity. Despite the limitations of modelling dengue disease in immunocompetent animals, infection of AnxA1 KO and FPR2 KO animals (and their respective littermates BALB/c and C57BL/6 mice) with high inoculum of DENV-2 was applied to investigate the role of endogenous AnxA1/FPR2 in dengue disease. Although viremia is not detectable in immunocompetent mice, we noticed transient viral RNA detection in the spleen starting 24 hr after infection, peaking at 48 hr or 72 hr, followed by disappearance at 96 hr, in line with previous studies using the same model (St John et al., 2013b; Syenina et al., 2015). Neither genetic deletion of AnxA1 or treatment with Ac_2-26 affected this profile. To complement our studies with immunocompetent animals mice, we performed experiments in A129 mice since they are known to be very susceptible to flaviviruses infections (de Freitas et al., 2019; Del Sarto et al., 2020) and develop a more severe disease manifestation that emulates several features of severe dengue in humans (Zandi et al., 2019). The later is considered a gold standard viremia model to test potential antiviral therapies (Meyts and Casanova, 2021; Williams et al., 2009). Using this model, we aimed to evaluate if the treatment with peptide Ac_2-26 has any impact on viral loads as well as in disease and inflammatory parameters. Of note, we were able to address the quantification of the viral loads by the plaque assay technique, the gold standard methodology to evaluate viable viral particles in vivo. Therefore, in our study we show, by two different techniques, that is, quantification of viral RNA (immunocompetent mice) and quantification of viable particles (A129 mice), and in two different models (high and low DENV inoculums) that neither absence of AnxA1 nor treatment with peptide Ac_2-26 interferes with the host ability to deal with the infection, despite its beneficial pro-resolving effects.

DENV infection initiates pro-inflammatory responses aiming to control virus spread that ultimately contributes to the immunopathology of dengue. For instance, MC activation in response to DENV plays an essential role in DENV-induced vascular pathology, particularly concerning the plasma leakage that causes hypovolemic shock in severe dengue (St John et al., 2013b; Syenina et al., 2015). This is supported by a correlation between circulating chymase levels and disease severity in humans (Rathore et al., 2020; Tissera et al., 2017). It has been recently described that AnxA1 acts as an endogenous modulator of MC degranulation in response to IgE/anti-IgE or compound 48/80, suggesting that this pro-resolving axis act as a brake in MC degranulation (Sinniah et al., 2016; Yazid et al., 2013). The present study confirmed that dengue disease is associated with increased plasma levels of MCPT-1 and identified enhanced secretion in animals lacking AnxA1 or FPR2/ALX. Herein, we have identified the ability of Ac_2-26 to reduce DENV-induced MC degranulation dose-dependently, a mechanism that might underpin the reduced MCPT-1 secretion and vascular dysfunction observed in AnxA1 peptide-treated animals. Together, in vivo and in vitro evidence suggest that Ac_2-26, at least in part, acts by attenuating MC degranulation evoked by DENV, protecting the host against vascular dysfunction associated with the disease. This mode of action points to this pathway as a relevant potential target for DENV infection treatment, as MCs are resistant to infection but play a key role in dengue pathogenesis (; Rathore et al., 2019; Syenina et al., 2015; Tissera et al., 2017).

Although no therapy is available for dengue disease beyond supportive care, recent studies have pointed towards a benefit of using corticoids in severe dengue with no significant adverse consequences or promotion of virus replication (Bandara and Herath, 2018; Bandara and Herath, 2020). Amongst the several genomic and non-genomic actions of glucocorticoids (GC), promotion of AnxA1 has shown to be relevant to the benefits afforded by steroids (De Caterina et al., 1993; Solito et al., 2003), as suggested by the loss of the therapeutic effect of GC on AnxA1 depleted animals (Patel et al., 2012; Perretti and D’Acquisto, 2009; Vago et al., 2012). Our study reveals a central role of the axis AnxA1-FPR2/ALX in the pathogenesis of dengue disease. We suggest that this critical anti-inflammatory/pro-resolving pathway is downregulated during dengue, contributing to unbalanced inflammation and the cytokine storm characteristic of the disease. Therefore, there is scope to hypothesise that the benefit of glucocorticoid in dengue disease could be partially attributed to promoting the glucocorticoid inducible Annexin A1-FPR2/ALX pathway. Further investigations should confirm AnxA1 as an underlying mechanism of GCs in dengue disease. The effectiveness of the treatment with AnxA1 peptide in our study suggests that GC therapy could be refined using a more targeted approach, particularly in infectious diseases where there is an underlying imbalance in the AnxA1 pathway. This opens an opportunity to explore small molecules targeting FPR2/ALX receptors, avoiding the spanning adverse consequences of glucocorticoids whilst targeting the defect in inflammation resolution operating in the disease settings. Further investigation on GC and GC-inducible AnxA1 is particularly relevant in the context of dengue disease, where the use of non-steroidal anti-inflammatory agents in dengue is discouraged, as they can increase the risk of bleeding (WHO, 2009).

Our results indicate that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in dengue disease. We show that inadequate engagement of resolution circuits contributes to the excessive inflammation observed in severe DENV infection. In addition, we provide evidence for the benefits of pharmacological therapy directed to modulating host immune responses in the absence of a direct antiviral effect. These findings point to a direction for future research on applying FPR2/ALX agonists as a therapeutic target for dengue disease. Since pathogenic immune responses are not exclusive to dengue but underlie the severity of several other viral diseases, including severe community-acquired pneumonia caused by viruses (D’Elia et al., 2013; Perrone et al., 2008) and COVID-19 (Jose and Manuel, 2020), our findings could be translated into different infectious settings, whereby targeting the AnxA1 pathway, with or without combination with antiviral drugs, holds promising therapeutic potential.

All data has been included in the manuscript, and source data files have been provided for Figures 1-6.

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Decision letter after peer review:

Thank you for submitting your work entitled "Targeting the Annexin A1-FPR2/ALX pathway for host-directed therapy in dengue disease" for further consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Frank Hirchhoff as Reviewing Editor and Reviewer #1, and the evaluation has been evaluated by Jos Van der Meer as Senior Editor. The reviewers have opted to remain anonymous.

There are some significant remaining issues that need to be addressed, as outlined below.

Essential revisions:

1. The improvements in disease symptoms appeared to be independent of the viral load. Since AC2-26 had previously been shown to have anti-inflammatory properties these findings are, while intriguing in the context of disease, not necessarily novel. The authors should mention that corticosteroids have been previously examined for the treatment of dengue disease and may exert beneficial effects through the induction of Annexin A1 and ALXR expression. They should also discuss why FPR2/ALX agonists may be better than treatment with corticosteroids to suppress exaggerated inflammation.

2. It is well established that DENV does not replicate efficiently in mice, but infection can occur using mouse-adapted strains of DENV and/or immunocompromised mouse strains. Here, the authors use a combination of wt (Balb/C and C57BL/6) and mouse mutant lines (including AG129 mice which have been shown to support robust infection with mouse adapted DENV strains) and infect them with a very high dose of DENV2 isolate. There are very limited viremia data shown throughout the manuscript which makes is somewhat difficult to interpret some of the results. How much are the effects in all the non-AG129 mouse lines attributable to an acute response to the high inoculum (and no/very limited replication) vs actual infection?

3. In Figure 4, when using IFNα/βR-/- mice (AG129), IL-6 and CCL5 instead of MCPT-1 and CCL2 were used as markers of systemic inflammatory mediators, which is not consistent with the previous experiments done in immunocompetent mice. No results of MCPT-1 and CCL2 changes in AG129 mice was presented and the authors didn't explain why they changed the markers in this manuscript. Since MCPT-1 is a mast cell-specific product, if it is not decreased in AG129 mice treated with Ac2-26 after DENV infection, how would the authors explain the protective effects of Ac2-26 in AG129 mice in the context of DENV infection?

4. The authors showed AnxA1 memetic peptide Ac2-26 could suppress MC degranulation. However, the same experiments should also be done in parallel in AnxA1-/- and FPR2/ALX-/- mice and in BMMCs derived from them. If Ac2-26 can prevent MC degranulation, it is expected to see decreased degranulation in AnxA1 mice but not FPR2/ALX KO mice when infected with DENV, both in vivo and in BMMCs. Finally, the peptide concentrations in mice (150 µg/animal) and in vitro (100 µM) are pretty high. The authors should mention whether any side effects were observed.

Reviewer #1 (Recommendations for the authors):

Dengue virus infection is spread by mosquitos and typically causes high fever, headache, vomiting, muscle and joint pains, and a characteristic skin rash. Typically, these symptoms resolved within a week but in some cases, dengue disease becomes more severe and is leads to hemorrhagic fever or dengue shock syndrome. Currently, no specific treatment against dengue disease is available. In the present study, Costa and colleagues hypothesized that excessive inflammatory responses play a key role in severe dengue disease and that anti-inflammatory molecules may be reduced in infected individuals. Specifically, they show that the circulating levels of anti-inflammatory Annexin A1 are reduced in both dengue patients and in infected mice. They further report that lack of Annexin A1 or its receptor FPR2/ALX enhance the disease in infected mice, while treatment with Annexin A1 agonistic peptide had beneficial effects. They further propose that the clinical outcome is dependent on inflammation and mast cell activation rather than the levels of viral replication. The authors conclude that Annexin A1 plays a key role in the clinical outcome of dengue virus infection and that FPR2/ALX agonists may offer therapeutic perspectives. Strength of the present study are that the authors analyzed both patient as well as mouse sample. Studies in knockout animals, provide strong evidence that the attenuating effect of Annexin A1 on the inflammatory response indeed attenuates the severity of dengue disease. Additional strengths are the evidence that an agonistic peptide exerts protective effect. A limitation is that the concentrations of the peptide used to monitor its effects (100 µM) are pretty high. In addition, the treatment is not specific and the concept of utilizing anti-inflammatory agents is not novel. Previous studies suggested that corticosteroids may prevent the progression to severe dengue disease (Rathnasiri Bandaraa and Herathb, Heliyon, 2018). Corticosteroids exert their anti-inflammatory effects by the induction of Annexin A1 and ALXR expression. This link should be mentioned and its needs to be explained why FPR2/ALX agonists may be better than treatment with corticosteroids to suppress exaggerated inflammation.

1. The authors should mention that corticosteroids have been previously examined for the treatment of dengue disease and may exert beneficial effects through the induction of Annexin A1 and ALXR expression. They should also discuss why FPR2/ALX agonists may be better than treatment with corticosteroids to suppress exaggerated inflammation.

2. Some references seem flawed (e.g. line 53, The Lancet Infectious, 2018). Should be checked throughout.

3. Line 254: "Groups were comparable for sex distribution". Table 1 shows that the proportion of females was higher in the control group and that, on average, age was higher in patients with severe dengue disease. This should be mentioned in the text.

4. It would be interesting to know whether the levels of Annexin A1 in individuals with dengue infection (Figure 1A) are inversely correlated with the levels of inflammatory cytokines.

5. The peptide concentrations in mice (150 µg/animal) and in vitro (100 µM) are pretty high. The authors should mention whether any side effects were observed.

6. The Annexin A1 levels in patients were about 10-fold lower than in mice (Figure 1). This difference should be discussed.

7. The authors mention that treatment with the Ac2-26 peptide did not affect systemic viral burden. It seems somewhat surprising that attenuation of innate immune activation doesn't affect the levels of viral replication. Please discuss.

Reviewer #2 (Recommendations for the authors):

In this manuscript, Costa et al., investigated the role of AnxA1-FPR2/ALX pathway in disease severity of dengue virus (DENV) infection and explores the therapeutic benefits of AnxA1 memetic peptide in DENV infection using mouse model. The authors show that serum levels of pro-resolving protein AnxA1 are significantly decreased in patients with DENV infection and infected mice. They show that the absence of AnxA-FPR2/ALX pathway in mice lead to aggravated dengue disease parameters by measuring hematological parameters, vascular leakage, and inflammatory factors. They further demonstrate that administration of a AxnA1 memetic peptide, Ac2-26, could improve these disease parameters in WT, AnxA1-/-, but not mice lacking the AnxA1 receptor, FPR2/ALX. In IFNα/βR-/- mice which are more susceptible to DENV infection, treatment of Ac2-26 improved the clinical outcome along with the disease parameters. The authors finally expore the mechanism of Ac2-26's protective effects and showing that Ac2-26 prevents mast cell degranulation both in vitro and in vivo models.

Overall, this study provides new insights on the mechanisms that underlie severe dengue disease. Notably, the data presented here suggest that the AnxA1-FPR2/ALX pathway may be a suitable potential target for host-directed therapy and a biomarker for stratifying the severity of in human dengue disease. The design of the experiments is largely adequate and has yielded data are mostly supportive of the conclusions drawn here.

Major issues that affect impact:

– The improvements in disease symptoms appeared to be independent of the viral load. Since AC2-26 had previously been shown to have anti-inflammatory properties these findings are, while intriguing in the context of disease, not necessarily novel.

– It is well established that DENV does not replicate efficiently in mice, but infection can occur using mouse-adapted strains of DENV and/or immunocompromised mouse strains. Here, the authors use a combination of wt (Balb/C and C57BL/6) and mouse mutant lines (including AG129 mice which have been shown to support robust infection with mouse adapted DENV strains) and infect them with a very high dose of DENV2 isolate. There are very limited viremia data shown throughout the manuscript which makes is somewhat difficult to interpret some of the results. How much are the effects in all the non-AG129 mouse lines attributable to an acute response to the high inoculum (and no/very limited replication) vs actual infection?

– In Figure 4, when using IFNα/βR-/- mice (AG129), IL-6 and CCL5 instead of MCPT-1 and CCL2 were used as markers of systemic inflammatory mediators, which is not consistent with the previous experiments done in immunocompetent mice. No results of MCPT-1 and CCL2 changes in AG129 mice was presented and the authors didn't explain why they changed the markers in this manuscript. Since MCPT-1 is a mast cell-specific product, if it is not decreased in AG129 mice treated with Ac2-26 after DENV infection, how would the authors explain the protective effects of Ac2-26 in AG129 mice in the context of DENV infection?

– The authors showed AnxA1 memetic peptide Ac2-26 could suppress MC degranulation. However, the same experiments should also be done in parallel in AnxA1-/- and FPR2/ALX-/- mice and in BMMCs derived from them. If Ac2-26 can prevent MC degranulation, it is expected to see decreased degranulation in AnxA1 mice but not FPR2/ALX KO mice when infected with DENV, both in vivo and in BMMCs.

Reviewer #3 (Recommendations for the authors):

Costa and colleagues investigated the involvement of the pro-resolving protein Annexin A1 (AnxA1) in controlling vascular leakage and cytokine storm associated with dengue disease most severe forms. They find a decrease of AnxA1 levels in the plasma of patients during acute dengue virus infection compared to healthy patients. This decrease is even more pronounced in patients with severe dengue, i.e. requiring hospitalization, compared to those with non-severe dengue. A similar AnxA1 profile was measured over time in dengue-infected wild-type mice of two different strains and further investigated in this model. Animals lacking AnxA1 or AnxA1-receptor FPR2/ALX showed more severe pathology than wild-type mice, while maintaining unchanged viral load. Importantly, the administration of an AnxA1 mimetic peptide strongly attenuated the manifestations of dengue disease in wild-type and AnxA1 knockout mice. This effect could be explained in part by the limitation of mast cell degranulation in vivo and in vitro.

The strength of the article lies in the combination of observations made in patients with reproducible phenotypes in different mouse models, as well as in the testing of molecules with therapeutic potential. The manuscript is scientifically sound and the experiments are well executed. In general, the data are of very good quality, well analyzed and clearly presented. This study provides a very interesting perspective on the pharmacology of resolution, highlighting the benefit of expanding the scope of therapies in the future beyond that of viral load control in order to limit the overshoot of immune responses and associated damage.

Essential revisions:

1. The improvements in disease symptoms appeared to be independent of the viral load. Since AC2-26 had previously been shown to have anti-inflammatory properties these findings are, while intriguing in the context of disease, not necessarily novel.

We have addressed this valid point in our reply to Reviewer #2 (comment #1). The relevance and novelty of our findings were further highlighted in the updated version of our manuscript.

The authors should mention that corticosteroids have been previously examined for the treatment of dengue disease and may exert beneficial effects through the induction of Annexin A1 and ALXR expression. They should also discuss why FPR2/ALX agonists may be better than treatment with corticosteroids to suppress exaggerated inflammation.

We have addressed this important point in our reply to Reviewer #1 (comment #1). Following the Reviewer's advice, the revised manuscript includes a discussion highlighting (i) the potential involvement of AnxA1 in the beneficial actions of corticosteroids in dengue disease and (ii) the therapeutic benefit of targeting this pathway over established anti-inflammatory therapies.

2. It is well established that DENV does not replicate efficiently in mice, but infection can occur using mouse-adapted strains of DENV and/or immunocompromised mouse strains. Here, the authors use a combination of wt (Balb/C and C57BL/6) and mouse mutant lines (including AG129 mice which have been shown to support robust infection with mouse adapted DENV strains) and infect them with a very high dose of DENV2 isolate. There are very limited viremia data shown throughout the manuscript which makes is somewhat difficult to interpret some of the results. How much are the effects in all the non-AG129 mouse lines attributable to an acute response to the high inoculum (and no/very limited replication) vs actual infection?

We have carefully addressed this concern in our reply to Reviewer #2 (comment #2) and included a new paragraph in the revised manuscript discussing this important point.

3. In Figure 4, when using IFNα/βR-/- mice (AG129), IL-6 and CCL5 instead of MCPT-1 and CCL2 were used as markers of systemic inflammatory mediators, which is not consistent with the previous experiments done in immunocompetent mice. No results of MCPT-1 and CCL2 changes in AG129 mice was presented and the authors didn't explain why they changed the markers in this manuscript. Since MCPT-1 is a mast cell-specific product, if it is not decreased in AG129 mice treated with Ac2-26 after DENV infection, how would the authors explain the protective effects of Ac2-26 in AG129 mice in the context of DENV infection?

As suggested by the Reviewer, we have included data on the systemic levels of MCPT-1 and CCL2 in A129 mice subjected to the model of severe dengue (Figure 4H,I). Please, refer to our reply to Reviewer #2 (comment #3).

4. The authors showed AnxA1 memetic peptide Ac2-26 could suppress MC degranulation. However, the same experiments should also be done in parallel in AnxA1-/- and FPR2/ALX-/- mice and in BMMCs derived from them. If Ac2-26 can prevent MC degranulation, it is expected to see decreased degranulation in AnxA1 mice but not FPR2/ALX KO mice when infected with DENV, both in vivo and in BMMCs.

As suggested by the Reviewer, we include new data in the revised version of the manuscript showing the dependence of FPR2/ALX for the modulatory effect of Ac_2-26 in mast cell degranulation (Figure 6B,C).

Finally, the peptide concentrations in mice (150 µg/animal) and in vitro (100 µM) are pretty high. The authors should mention whether any side effects were observed.

As detailed in our reply to Reviewer #1 (comment #5), the in vivo dose used in our study was based on previous publications from our group and others (Damazo, Yona, Flower, Perretti, and Oliani, 2006; Galvao et al., 2017; Machado et al., 2020; Perretti, Ahluwalia, Harris, Goulding, and Flower, 1993; Vago et al., 2012). Using 150 µg/animal, we did not observe any notable adverse effects in our experiments (we have included this observation in the revised text).

Our in vitro concentration of Ac_2-26 is consistent with previous studies on mast cells, where the prevention of mast cell degranulation is only achieved with high peptide concentrations (M. P. Oliveira, Prates, Gimenes, Correa, and Oliani, 2021). Besides, the concentration of Ac_2-26 used in our in vitro studies was based on a concentration-response performed in bone-marrow-derived mast cells (Figure 6D of the updated manuscript).

Reviewer #1 (Recommendations for the authors):

1. The authors should mention that corticosteroids have been previously examined for the treatment of dengue disease and may exert beneficial effects through the induction of Annexin A1 and ALXR expression. They should also discuss why FPR2/ALX agonists may be better than treatment with corticosteroids to suppress exaggerated inflammation.

We thank the Reviewer for their insightful comment. Although guidelines from the World Health Organization for managing dengue fever only provide recommendations on fluid management and supportive care (WHO, ‎2009), the use of anti-inflammatories in dengue disease has been evaluated. While the use of non-steroidal anti-inflammatory agents in dengue is discouraged, as they can increase the risk of bleeding (PAHO, 2016; WHO, ‎2009), recent studies have pointed towards a benefit of using corticoids in severe dengue with no significant adverse consequences or promotion of virus replication (S. M. R. Bandara and Herath, 2018; S. M. Rathnasiri Bandara and Herath, 2020). Noteworthy, amongst the several genomic and non-genomic actions of glucocorticoids, promotion of AnxA1 has shown to be relevant to the benefits afforded by steroids (De Caterina et al., 1993; Solito et al., 2003), as suggested by the loss of the therapeutic effect of glucocorticoids on AnxA1 depleted animals (Damazo et al., 2006; Patel et al., 2012; Perretti and D'Acquisto, 2009; Vago et al., 2012).

Our study reveals a central role of the axis AnxA1-FPR2/ALX in the pathogenesis of dengue disease. We suggest that this critical anti-inflammatory/pro-resolving pathway is downregulated during dengue, contributing to unbalanced inflammation and the cytokine storm characteristic of the disease. Therefore, there is scope to hypothesise that the benefit of corticosteroids in dengue disease could be partially attributed to promoting the glucocorticoid-inducible Annexin A1/FPR2/ALX pathway. Further investigations should confirm AnxA1 as an underlying mechanism of corticosteroids in dengue disease.

The effectiveness of the treatment with AnxA1 peptide in our study suggests that glucocorticoid therapy could be refined using a more targeted approach, particularly in infectious diseases where there is an underlying imbalance in the AnxA1 pathway. This opens an opportunity to explore small molecules targeting FPR2/ALX receptors, avoiding the spanning adverse consequences of glucocorticoids while targeting the defect in inflammation resolution operating in the disease settings. Further investigation on GC and GC-inducible AnxA1 is particularly relevant in dengue disease, where non-steroidal anti-inflammatory agents are discouraged, as mentioned above.

The revised manuscript includes a discussion highlighting (i) the potential involvement of AnxA1 in the beneficial actions of corticosteroids in dengue disease and (ii) the therapeutic benefit of targeting this pathway over established anti-inflammatory therapies (lines 620-640; 645).

2. Some references seem flawed (e.g. line 53, The Lancet Infectious, 2018). Should be checked throughout.

Thank you for noting this. We have replaced the reference in line 53 and thoroughly revised the other references in the manuscript.

3. Line 254: "Groups were comparable for sex distribution". Table 1 shows that the proportion of females was higher in the control group and that, on average, age was higher in patients with severe dengue disease. This should be mentioned in the text.

Thank you for your comment. Higher age in severe dengue cases is expected since older age is a risk factor for progression to severe disease (Rowe et al., 2014; Sangkaew et al., 2021). We have highlighted this age distribution in the updated version of our manuscript (line 263). As depicted in Table 1 legend, the sex ratio was statistically similar between the groups (p>0.1), making the groups comparable for sex distribution.

4. It would be interesting to know whether the levels of Annexin A1 in individuals with dengue infection (Figure 1A) are inversely correlated with the levels of inflammatory cytokines.

Although we agree with the Reviewer that this could be an interesting point to be addressed, we do not hold additional plasma samples to perform further ELISA assays. However, our research group is conducting an in-depth analysis of imbalances between inflammatory and pro-resolving mediators in plasma samples from dengue patients that will be comprehensively covered in a future publication.

Of note, in both models of dengue disease (immunocompetent and IFNα/βR KO animals), we show that decreased levels of AnxA1 during DENV infection are mirrored by higher levels of inflammatory markers, including the mast cell-specific product.

5. The peptide concentrations in mice (150 µg/animal) and in vitro (100 µM) are pretty high. The authors should mention whether any side effects were observed.

The dose of Ac_2-26 peptide (150μg/animal) was based on previous publications, such as those in the settings of peritonitis (Damazo et al., 2006), gout (Galvao et al., 2017), air pouch (Perretti et al., 1993), pleurisy (Vago et al., 2012) and lung infection (Machado et al., 2020). Initial studies on Ac_2-26 have used a dose of 200 µg per animal to prevent leukocyte migration induced by IL-1β, with no reports of adverse effects (Perretti et al., 1993). In addition, we did not observe any notable adverse effect in our experiments, as mentioned in line 390 of the updated manuscript.

Our in vitro concentration of Ac_2-26 is consistent with previous studies on mast cells (cell lines, BMMC and subcutaneous tissue fragments), where prevention of mast cell degranulation is only achieved with a concentration in the order of micrograms per millilitre (M. P. Oliveira et al., 2021). To confirm the requirement of a high concentration of Ac_2-26 in vitro, we performed a concentration-response, as depicted in Figure 6D of the updated manuscript.

6. The Annexin A1 levels in patients were about 10-fold lower than in mice (Figure 1). This difference should be discussed.

We thank the Reviewer for their comment. The plasma levels observed in our study are in line with previous measurements performed with a variety of studies using homemade ELISA assays or commercial ELISA kits (Adel et al., 2020; Purvis et al., 2019; Santana et al., 2021). Significant differences between animal and human samples in the same study were noticed before (Senchenkova et al., 2019; Xu et al., 2021). We have added this discussion in line 284 of the manuscript.

7. The authors mention that treatment with the Ac2-26 peptide did not affect systemic viral burden. It seems somewhat surprising that attenuation of innate immune activation doesn't affect the levels of viral replication. Please discuss.

This is an important point that has been discussed throughout the manuscript and further highlighted in the revised document (lines 89; 623; 648).

The ability of pro-resolving drugs to balance inflammation without hampering the host's ability to deal with pathogens is, in fact, a hallmark of resolution pharmacology, representing an advantage compared to standard anti-inflammatory drugs (Perretti, Leroy, Bland, and Montero-Melendez, 2015).

Several studies have suggested that pro-resolving mediators can suppress inflammation and diminish inflammatory responses while improving the phagocytosis of pathogens. This was initially demonstrated for specialised lipid mediators in bacterial infection (Chiang et al., 2012; Decker, Sadhu, and Fredman, 2021; Sekheri, El Kebir, Edner, and Filep, 2020), with subsequent studies with Annexin A1 on various infection models (Gobbetti et al., 2014; Machado et al., 2020; L. G. Oliveira et al., 2017; Tzelepis et al., 2015; Vanessa et al., 2015). Therefore, it is already well accepted that pro-resolving drugs can promote innate immunity against bacterial pathogens while reducing the deleterious effects of inflammation. Here we translate these findings into a model of infection. Please refer to our reply to Reviewer #2 (comment 1), where we discuss the novelty of our study.

Reviewer #2 (Recommendations for the authors):

Major issues that affect impact:

– The improvements in disease symptoms appeared to be independent of the viral load. Since AC2-26 had previously been shown to have anti-inflammatory properties these findings are, while intriguing in the context of disease, not necessarily novel.

Thank you for your comment. As discussed through the manuscript, the protective effect of AnxA1 and its mimetic peptide have been mainly demonstrated in sterile settings (e.g. inflammation triggered by LPS, uric acid crystals, DSS, and atherosclerosis genetically engineered animals), revealing various anti-inflammatory and pro-resolving actions of these molecules, including the promotion of granulocyte apoptosis, efferocytosis, and macrophage reprogramming (Fredman et al., 2015; Galvao et al., 2017; Gimenes et al., 2015; Kusters et al., 2015; Leoni et al., 2015; Locatelli et al., 2014; Sugimoto, Vago, Perretti, and Teixeira, 2019). Later, studies suggested that pro-resolving mediators can suppress inflammation and reduce the inflammatory response while improving the phagocytosis of pathogens. This was initially demonstrated for specialised lipid mediators in bacterial infection (Chiang et al., 2012; Decker et al., 2021; Sekheri et al., 2020), with subsequent studies with Annexin A1 on various infection models (Gobbetti et al., 2014; Machado et al., 2020; L. G. Oliveira et al., 2017; Tzelepis et al., 2015; Vanessa et al., 2015).

However, data on the role of resolving mediators on viral infections are scarce, with most current studies focusing on lipidic mediators of resolution (Walker et al., 2021) and insufficient data on the role of Annexin A1 in viral diseases (Schloer et al., 2019). Findings that Annexin A1 could promote influenza A virus replication in vitro were particularly surprising (Arora et al., 2016). Therefore, while studies on bacterial infection consistently revealed the ability of pro-resolving drugs to facilitate innate immune responses against the pathogen while reducing the deleterious effects of inflammation, the translational potential of these results to viral infection was less clear.

Our work is the first to access the protective role of AnxA1 in dengue infection and to reveal the therapeutic potential of Annexin A1 mimetic peptides. We show that AnxA1 can suppress the inflammatory response without hampering the protective immunity against the virus. Besides, although previous studies have suggested that AnxA1 could suppress mast cell degranulation (Gimenes et al., 2015; M. P. Oliveira et al., 2021; Parisi, Correa, and Gil, 2019; Stuqui et al., 2015), our manuscript is the first to describe the role of AnxA1 on mast cells degranulation triggered by DENV.

Finally, our findings are relevant since pathogenic immune responses are not exclusive to dengue but underlie the severity of several other viral diseases, including severe community-acquired pneumonia caused by viruses (D'Elia, Harrison, Oyston, Lukaszewski, and Clark, 2013; Perrone, Plowden, Garcia-Sastre, Katz, and Tumpey, 2008) and COVID-19 (Jose and Manuel, 2020). Therefore, other researchers could explore our data and translate it into different infectious settings, whereby targeting the AnxA1 pathway, with or without combination with antiviral drugs, holds promising therapeutic potential.

The relevance and novelty of our findings were further highlighted in the updated version of our manuscript (lines 89; 623; 648).

– It is well established that DENV does not replicate efficiently in mice, but infection can occur using mouse-adapted strains of DENV and/or immunocompromised mouse strains. Here, the authors use a combination of wt (Balb/C and C57BL/6) and mouse mutant lines (including AG129 mice which have been shown to support robust infection with mouse adapted DENV strains) and infect them with a very high dose of DENV2 isolate. There are very limited viremia data shown throughout the manuscript which makes is somewhat difficult to interpret some of the results. How much are the effects in all the non-AG129 mouse lines attributable to an acute response to the high inoculum (and no/very limited replication) vs actual infection?

Despite the limitations of modelling dengue disease in immunocompetent animals, in our study, AnxA1 and FPR2/ALX knockout animals (and their respective littermates BALB/c and C57BL/6 mice) were used as a genetic tool to investigate the role of endogenous AnxA1 in dengue disease. As mentioned by the Reviewer, DENV low passage clinical isolates do not replicate efficiently in immunocompetent mice, and for that reason, the inoculum must be higher than when using adapted strains to induce a similar disease profile. Of note, here we used 1x10⁶ PFU of DENV-2 per mouse (for immunocompetent strains): this is the same virus strain and inoculum used in recent publications (Rathore et al., 2019; St John, Rathore, Raghavan, Ng, and Abraham, 2013). Although viremia is not detectable in immunocompetent mice (St John et al., 2013), we noticed transient viral RNA detection in the spleen starting 24h after infection, peaking at 72h, followed by disappearance at 96h (Figure 2_supplement 1 and Figure 3_supplement 1). These data are in line with those presented in previous studies using the same model (St John et al., 2013; Syenina, Jagaraj, Aman, Sridharan, and St John, 2015).

Although resistant to DENV infection (Shresta et al., 2004; Yauch and Shresta, 2008), it has been reported that infection of immunocompetent mice with DENV can induce a self-limited disease, like dengue fever. While limited by the lack of detectable viremia, these models allow the study of critical immune components, such as mast cells (Rathore et al., 2019; St John et al., 2013; Syenina et al., 2015), and their contribution to the classical haematological abnormalities and altered vascular permeability observed during DENV infection (Assuncao-Miranda et al., 2010; Chen, Hofman, Kung, Lin, and Wu-Hsieh, 2007).

To complement our studies with Balb/c and C57Bl/6 mice, we have performed experiments in A129 mice since they are known to be highly susceptible to flaviviruses infections (de Freitas et al., 2019; Del Sarto et al., 2020) and develop a more severe disease manifestation that emulates several features of severe dengue in humans (Zandi et al., 2019). This model is considered a gold standard viremia model to test potential antiviral therapies (Meyts and Casanova, 2021; Williams, Zompi, Beatty, and Harris, 2009). Using this model, we aimed to evaluate if the treatment with peptide Ac_2-26 impacts viral loads, besides the alterations on disease and inflammatory parameters observed in immunocompetent animals. We have successfully recovered viable virus from samples using the plaque assay technique, as described in the Method section of the manuscript. Since A129 animals are more susceptible to infection, we could use a lower inoculum of the same non-adapted strain in this model (10³ PFU).

Therefore, we have applied two different techniques (quantification of viral RNA for immunocompetent mice and quantification of viable particles for A129 KO mice) and two different models (high and low DENV inoculums) to investigate the impact of endogenous and exogenous AnxA1 on the host ability to deal with the infection.

We have included a new paragraph in the revised manuscript discussing this important point (lines 586-605).

– In Figure 4, when using IFNα/βR-/- mice (AG129), IL-6 and CCL5 instead of MCPT-1 and CCL2 were used as markers of systemic inflammatory mediators, which is not consistent with the previous experiments done in immunocompetent mice. No results of MCPT-1 and CCL2 changes in AG129 mice was presented and the authors didn't explain why they changed the markers in this manuscript. Since MCPT-1 is a mast cell-specific product, if it is not decreased in AG129 mice treated with Ac2-26 after DENV infection, how would the authors explain the protective effects of Ac2-26 in AG129 mice in the context of DENV infection?

Thank you for this important comment. The inflammatory markers investigated in the model of severe dengue (A129 animals) were selected based on previous publications showing CCL5 and IL6 as classical inflammatory markers in dengue and a requirement of CCR5 for DENV replication and infection development (Guabiraba et al., 2010; Marques et al., 2015). Besides, liver injury, a feature of dengue disease observed in our A129 model (Binh, Matheus, Huong, Deparis, and Marechal, 2009; Trung et al., 2010) seems to correlate with high levels of CCL5 (Conceicao et al., 2010; Suksanpaisan, Cabrera-Hernandez, and Smith, 2007),

As suggested by the Reviewer, we have included data on the systemic levels of MCPT-1 and CCL2 in A129 mice subjected to the model of severe dengue (Figure 4H-J; line 383). As observed in immunocompetent mice, Ac_2-26 attenuated the systemic release of the mast cell chymase MCPT-1 induced by DENV-2 (Figure 4H), although it did not affect systemic levels of CCL2 (Figure 4I). The lack of effect of Ac_2-26 in CCL2 levels might be justified by the higher severity of the model in A129 animals, resulting in significantly higher production of CCL2 compared to immunocompetent animals infected with DENV (mean ± SD CCL2 production at the peak of 585.0 ± 61.9 for C57BL/6; 621.2 ± 100.5 for BALB/c; and 977.8 ± 110.7 for A129 mice).

– The authors showed AnxA1 memetic peptide Ac2-26 could suppress MC degranulation. However, the same experiments should also be done in parallel in AnxA1-/- and FPR2/ALX-/- mice and in BMMCs derived from them. If Ac2-26 can prevent MC degranulation, it is expected to see decreased degranulation in AnxA1 mice but not FPR2/ALX KO mice when infected with DENV, both in vivo and in BMMCs.

Thank you for your suggestion. We have included new data in the revised version of the manuscript showing the dependence of FPR2/ALX for the modulatory effect of Ac_2-26 in mast cell degranulation (Figure 6B,C; methods lines 201-207; results lines 461-469).

As predicted by the Reviewer, we verified that systemic administration of Ac_2-26 successfully reduced mast cell degranulation in AnxA1 KO mice (Figure 6B), but failed to rescue DENV-2 induced degranulation in FPR2/ALX KO animals (Figure 6C). Notably, the absence of FPR2/ALX significantly increased mast cell degranulation in response to the local challenge with DENV compared to the WT counterparts (Figure 6C).

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Author details

1. Vivian Vasconcelos Costa

   Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft

   Contributed equally with

   Michelle A Sugimoto

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0175-642X

2. Michelle A Sugimoto

   1. Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
   2. School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
   3. William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft

   Contributed equally with

   Vivian Vasconcelos Costa

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4527-6065

3. Josy Hubner

   Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Caio S Bonilha

   1. Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
   2. Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom

   Contribution

   Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

5. Celso Martins Queiroz-Junior

   Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Contribution

   Data curation, Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

6. Marcela Helena Gonçalves-Pereira

   Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Contribution

   Data curation, Investigation, Resources, Writing - review and editing

   Competing interests

   No competing interests declared

7. Jianmin Chen

   William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

8. Thomas Gobbetti

   William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Gisele Olinto Libanio Rodrigues

   Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

10. Jordana L Bambirra

    Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

11. Ingredy B Passos

    Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Data curation, Investigation, Methodology

    Competing interests

    No competing interests declared

12. Carla Elizabeth Machado Lopes

    Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Investigation

    Competing interests

    No competing interests declared

13. Thaiane P Moreira

    Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Investigation

    Competing interests

    No competing interests declared

14. Kennedy Bonjour

    Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil

    Contribution

    Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - review and editing

    Competing interests

    No competing interests declared

15. Rossana CN Melo

    Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil

    Contribution

    Data curation, Investigation, Methodology

    Competing interests

    No competing interests declared

16. Milton AP Oliveira

    Tropical Pathology and Public Health Institute, Universidade Federal de Goiás, Goiânia, Brazil

    Contribution

    Resources

    Competing interests

    No competing interests declared

17. Marcus Vinicius M Andrade

    School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Methodology, Resources, Supervision

    Competing interests

    No competing interests declared

18. Lirlândia Pires Sousa

    Department of Clinical and Toxicological Analyses, School of Pharmacy, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Investigation, Methodology, Resources

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1042-9762

19. Danielle Gloria Souza

    Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Conceptualization, Investigation, Methodology, Supervision, Visualization

    Competing interests

    No competing interests declared

20. Helton da Costa Santiago

    Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Visualization

    Competing interests

    No competing interests declared

21. Mauro Perretti

    1. William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
    2. Centre for Inflammation and Therapeutic Innovation, Queen Mary University of London, London, United Kingdom

    Contribution

    Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft

    Contributed equally with

    Mauro Martins Teixeira

    For correspondence

    m.perretti@qmul.ac.uk

    Competing interests

    is on the Scientific Advisory Board of ResoTher Pharma AS, which is interested in developing Annexin A1-derived peptides for cardiovascular settings

    "This ORCID iD identifies the author of this article:" 0000-0003-2068-3331

22. Mauro Martins Teixeira

    Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Contribution

    Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft

    Contributed equally with

    Mauro Perretti

    For correspondence

    mmtex.ufmg@gmail.com

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6944-3008

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