Targeting the Annexin A1-FPR2/ALX pathway for host-directed therapy in dengue disease

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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