Treatment with lipoxin A 4 improves influenza A infection outcome through macrophage reprogramming, anti-inflammatory and pro-resolutive responses

Objective and design: Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. Treatment: Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 mg/kg/day, i.p.) at day 3 post-infection. Methods Mortality rate was assessed up to day 21 and inflammatory parameters were assessed at days 5 and 7. Results AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in FPR2/3 −/− animals. In mice treated with LXA4 (50mg/kg/day, i.p., days 3–6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of anti-inflammatory T cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. Conclusions Therefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.


INTRODUCTION
In uenza A virus (IAV) is a virus from the Orthomyxoviridae family responsible for high levels of mortality and morbidity worldwide 1 and the leading pathogen of many outbreaks and pandemics such as Spanish Flu (1918), Asian Flu (1957), Hong Kong Flu (1968) and Swine Flu (2009) 2,3 .The World Health Organization estimates one billion cases of IAV infection per year with 3-5 million severe cases and 300.000-500.000deaths 4 .Although there are vaccines and anti-viral drugs used for the treatment of in uenza infection 5 , due to the high level of mortality and morbidity, nding additional strategies to minimize the impact of this infection on public health is greatly necessary.
In uenza virus infects the upper and lower respiratory tract and can reach the alveolar epithelium and alveolar macrophages 6 .Binding between the sialic acid on the surface of host cells and viral hemagglutinin 7 triggers the death of infected cells by apoptosis and necroptosis 8,9 .The activation of apoptosis of infected cells is important to control viral replication and, consequently, decreases viral spread specially when the virus escapes the immune system 10 .Once infected, cells recognize pathogen associated molecules patterns (PAMPs) through the activation of toll like receptors (TLRs) inducing not only the recruitment of immune cells to the site of infection, but also the production of cytokines such as interleukin (IL)-1β, IL-6 and interferons (IFNs) [10][11][12][13] .Macrophages and neutrophils are the most recruited cells into the infected lungs during IAV infection 14,15 .In the beginning of infection, macrophages are classically activated expressing markers such as iNOS and MHCII, along with cytokines and neutrophil extracellular traps (NETs), they are crucial to prevent viral replication 16,17 .
Host mediated immune pathology is a critical contributor to lung injury during IAV infection.A better outcome of infection relies on the activation of the resolution phase of in ammation in which antiin ammatory cytokines and pro-resolving mediators are produced 18 .We have previously shown that treatment with certain pro-resolving molecules is bene cial in the course of viral infections, including dengue virus, chikungunya virus and IAV [19][20][21] .For example, treatment with annexin-A1 greatly prevented disease severity after dengue virus infection and shortened chronicity in a model of chikungunya virus infection 20 .Similarly, treatment with angiotensin 1-7 (Angio 1-7), a pro-resolving molecule, signi cantly decreased pulmonary in ammation and damage associated with IAV infection in mice, an effect associated with fewer animals succumbing to infection 21 .These studies have led to the suggestion that pro-resolving molecules may be an useful adjunct therapy for the treatment of infectious diseases 22 .
Lipoxin A 4 (LXA 4 ) is an eicosanoid that promotes the resolution of in ammation through pleiotropic responses, are mediated via Formyl Peptide Receptor 2 (FPR2), a GPCR (designated FPR2/3 in mice).Chemical instability and complex synthesis have limited exploitation of the therapeutic potential of the lipoxins.We have developed several synthetic lipoxins mimetics where the triene core of LXA 4 has been replaced by heteroaromatic moieties with enhanced chemical stability, such as AT-01-KG, which contains a dimethylimidazole core 23,24 .The LXA 4 analogue AT-01-KG, a dimethyl-imidazole-containing lipoxin A 4 analogue, has both classic anti-in ammatory effects by reducing neutrophil in ux and pro-in ammatory cytokines but also pro-resolving effects 25 .In the current study, we evaluated whether treatment with AT-01-KG would favorably modify the course of IAV infection in mice.Mechanisms associated with protection were also evaluated.

Mice
Male wild-type and FPR2/3 -/-(KO) C57BL/6J mice (8-12 weeks old), weighing 18-25g, were obtained from the Central Animal Facility from Universidade Federal de Minas Gerais (UFMG/Brazil) and from the Animal Facility from Instituto de Ciências Biológicas in UFMG/Brazil, respectively.Mice were randomized by sex and assigned to experimental groups.They were maintained with free access to commercial chow and water in a 12-h dark-light cycle in the thermoneutral zone for mice.All procedures described had prior approval of the local animal ethics committee (CETEA/ UFMG 65/2021).WT C57BL/6 (6 to 8week-old) mice were also purchased from Jackson Laboratory (Bar Harbor, Maine) and colonies were subsequently maintained under speci c pathogen-free conditions at UPMC Children's Hospital of Pittsburgh.All animals' procedures described here had prior approval of the animal ethics committee of the Federal University of Minas Gerais and the University of Pittsburgh Institutional Animal Care and Use Committee.

IAV infection
The in uenza strains used in the study were in uenza A WSN/33 H1N1 produced in chicken eggs and then co-cultured with MDCK cells as previous described 26 and in uenza A/PR/8/34 (in uenza H1N1) propagated in chicken eggs as described 27 .Animals were anesthetized with ketamine/xylazine and received 10 4 PFU (lethal dose 50%) as previous described 3 .
Treatments started at 3 days post-infection and euthanized at days 5 and 7 or maintained up to 21 days post initial infection 29,30

Assessment of lung In ammation
The trachea was exposed, and bronchoalveolar lavage (BAL) was performed with two aliquots containing 1mL of sterile PBS.Saline solution was injected and collected three times into the lungs using a cannula and syringe.Then the BAL uid (BALF) samples were centrifuged at 600 x g for 10 min at 4 • C to count leukocytes.In addition, cytospin (Shandon III) of these leukocytes was performed for differential counting on slides stained with May-Grunwald-Giemsa, based on morphological criteria.Each slide was counted three times and the percentage was used to calculate the absolute number of each type of leukocyte, as previously described 31,32 .The right upper lobe of the lungs was mechanically homogenized in sterile PBS in order to measure cytokine production by Bio-plex assay (Bio-Rad, Hercules, CA) and the right middle and lower lobes were used for ow cytometry.

Assessment of Bone Marrow-Derived Macrophage (BMDM) efferocytosis
Bone marrow cells were sterilely isolated from the femurs and tibias of mice, and grown for 7 days in complete DMEM media supplemented with 20ng/mL GM-CSF (PeproTech, NJ), as previously described 33 .On day 6, adherent cells (BMDMs) were recovered using a cell scraper (Genemate, Kaysville, UT) with gentle mechanical scraping.Bone marrow derived cells were plated to 2.5 × 10 5 cells/mL in 300ul of media in 96 well tissue culture-treated plates then rested overnight in complete DMEM media supplemented with 20ng/mL GM-CSF (PeproTech, NJ).BMDMs were infected with 0.1 MOI of in uenza A WSN/33 H1N1 for 30min.Then, wells were washed with PBS 1% and complete DMEM was added with or without 1nM of AT-01-KG for 24 hours.On the following day CFSE labeled apoptotic thymocytes were generated as described 34 and co-cultured with BMDMs for 1 hour at 37°C, at a ratio of 1:3.Finally, cells were super cially stained by labeled anti-F4/80 (PE-Cy7-eBioscience). Assessment of efferocytosis was performed by ow cytometry for F4/80 + /CFSE + cells.

Assessment of Leukocyte Apoptosis and Efferocytosis
Apoptosis and efferocytosis was assessed morphologically, as previously described 26,35 .BALF was collected 7 days post IAV infection.Cells were centrifuged, xed, and stained with May-Grünwald-Giemsa and counted using oil immersion microscopy (100 objective) to determine the proportion of cells with distinctive apoptotic morphology (cells presenting chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies, out or inside macrophages) and macrophage undergoing efferocytosis.At least 300 cells were counted/slide.Efferocytosis was also assessed by ow cytometry considering cells F480+/CSFE+.

Flow Cytometry analysis
For in vivo experiments, mouse lungs were aseptically dissected into small sections, digested for 1 hour at 37°C in 1mg/mL collagenase media, and ltered with 70µm lters 36 .Two panels were made for myeloid and lymphoid cell staining.After live/dead staining with LIVE/DEAD blue or Zombie NIR, extracellular staining was done at 4°C for 30 minutes.Single cell suspensions staining is shown (Supplementary Fig. 4).The gating strategy used with FlowJo (Tree Star, Ashland, OR) is shown (Supplementary Figs. 2 and 3).Dimensionality reduction t-SNE analysis followed by FlowSOM clustering analysis was performed using Cytobank (Beckman Coulter, Indianapolis, IN).For in vitro experiments, cells from BAL were stained by labeled antibodies anti-F4/80 (25-4801-82, PEcy7, eBioscience, USA).

Histopathology assessment
After the reperfusion of the lungs, the left lung was collected and xed in 4% neutral phosphate-buffered formalin (pH 7.4), as described previously 37 .Tissues were dehydrated gradually in ethanol, embedded in para n, cut into 4mm sections, stained with hematoxylin-eosin, and analyzed under light microscopy by a pathologist blinded to the treatments.Lung structure, in ammation, edema and hemorrhage were quanti ed using a semi-quantitative score with increasing severity of changes (0, absent; 1, minimal; 2, slight; 3, moderate; 4, marked; and 5, severe).All parameters were included in an overall 20 points score.

Statistical Analysis
Data were analyzed using GraphPad Prism (Boston, MA) software.Experiments were repeated twice.All data are presented as mean ± SEM.Signi cance was determined by unpaired Student's t test or one-way ANOVA followed by post-hoc Bonferroni or Tukey's test for multiple comparisons.Area under the curve were used for mortality data analysis followed by a one-way ANOVA test.

Treatment with AT-01-KG improved survival, lung damage and reduced cell in ltration
LXA 4 is a specialized pro-resolving mediator that contributes to control of in ammation, reducing lung damage 38 .AT-01-KG is a synthetic lipoxin-A4 mimetic where the triene core of LXA 4 is replaced by a heteroaromatic imidazole.In order to analyze the potential effect of AT-01-KG as a potential treatment for IAV infection, we analyzed three distinct groups: PBS-treated uninfected mice (Mock), vehicle treated IAV infected mice (Veh.) and AT-01-KG treated IAV infected mice (AT-01-KG).Daily treatment was performed intraperitonially (ip) starting on day 3 post-infection up to the day of evaluation.Vehicle and AT-01-KG groups were infected with H1N1 IAV and in ammatory parameters were evaluated 5 and 7 days post-infection along with a survival curve (Fig. 1A).Mice treated with AT-01-KG showed improved survival (Fig. 1B).As anticipated, IAV infection was associated with increased protein in BALF (Fig. 1C).
The increase protein in BALF was signi cantly reduced in AT-01-KG treated mice 5 days post infection relative to vehicle treated mice.Histopathology analysis showed increased in ammation and damage in the lungs of untreated mice compared to the mock group (Figs.1D and E).However, decreased in ammation and damage were seen in the lungs of treated mice compared to untreated mice (Figs.1F   and G).
Even though the treatment did not affect the ability of the host to control viral replication (Supplementary Fig. 1A), BALF analysis showed reduced cell in ltration induced by the AT-01-KG treatment (Fig. 2A), particularly reduced numbers of mononuclear cells (Fig. 2B) and neutrophils (Fig. 2C) on day 7 postinfection.Thus, we decided to further investigate the pathology 7 post-infection in vehicle-treated and LXA4-treated mice.As anticipated, AT-01-KG treatment did not impact the number of cells in the mock group (Supplementary Fig. 1B).

LXA 4 treatment induced macrophage reprogramming towards an alternatively activated phenotype
Macrophage reprogramming from a classically activated phenotype towards an alternatively activated phenotype is a crucial step in the resolution phase of in ammation to control pro-in ammatory response contributing to an anti-in ammatory and pro-resolutive environment 39 .Specialized pro-resolving mediators, such as LXA 4 , are known to be able to induce macrophage reprogramming during resolution 38 .Flow cytometry analysis showed that LXA 4 treatment not only increased the number of macrophages in the lungs of treated mice (Fig. 3A and Supplementary Fig. 2) but also decreased frequency of macrophages expressing classically activated markers such as iNOS and MHCII (Fig. 3B).
The treatment was also associate with increased frequency of macrophages expressing alternatively activated markers such as Arg1 and CD206 (Fig. 3C).Macrophages subpopulation analysis showed that infected mice presented increased frequency of exudate macrophages (CD24-CD64 + CD11b + CD11c+) (Fig. 3D) and interstitial macrophages (CD24-CD64 + CD11b + CD11c-) (Fig. 3G), however, reduced the frequency of alveolar macrophages (Supplementary Fig. 1C).The treatment reduced the frequency of exudate and interstitial macrophages expressing classically activated markers (Figs. 3E and H) and increased interstitial macrophages expressing alternatively activated markers (Figure I).There was a trend towards an increase in the expression of alternatively activated markers in exudate macrophages (Fig. 3F).Alveolar macrophages from infected mice did not show signi cantly increased expression of iNOS, MHCII, Arg1 and CD206 following IAV infection preventing the analysis of macrophage reprogramming in this population (data not shown).

Frequency of apoptotic cells and macrophage efferocytosis was increased by AT-01-KG treatment
The activation of apoptosis in neutrophils followed by their efferocytosis by alternatively activated macrophages are key to immune cell clearance which contributes to the return to homeostasis 40 .Therefore, aiming to determine if AT-01-KG treatment induces apoptosis and efferocytosis, we rst tested that hypothesis in vitro.BMDMs were infected with 0.1 MOI of HIN1 for 30min then treated with vehicle or 1nM of AT-01-KG for 24h.CFSE labeled apoptotic cells were then added for 1 hour in order to analyze efferocytosis by ow cytometry measuring F480 + /CFSE + cells.Treated cells presented a high frequency of macrophage efferocytosis compared to untreated cells (Fig. 4A).In vivo, treated mice also presented increased frequency of apoptotic cells (Fig. 4B and C) and macrophage efferocytosis (Fig. 4D   and E) in the BALF, as assessed by morphology.

Levels of pro-in ammatory cytokines are decreased in the lungs of LXA 4 -treated mice
Since persistent cytokine production is associated with greater tissue damage and mortality 41 and LXA 4 has been shown to control levels of pro-in ammatory cytokine 42 we aimed to assess the cytokine pro le in the lungs of the mice through a Bio-plex 23-plex assay.A heat map (Fig. 5A) of all cytokines measured showed different cytokine pro les in the lungs of mice for each condition.Pro-in ammatory mediators such as G-CSF, IFNγ and IL-6 increases susceptibility to IAV lung injury 43 and contributes to elevated neutrophil in ux 44 .We showed that treatment with LXA 4 was able to decrease levels of those mediators (Figs.5B-D).Interestingly, treated mice had increased levels of IL-12p40 in the lungs.

Treatment with LXA 4 increased anti-in ammatory and regulatory T cells
Lymphoid cells play an important role controlling IAV infection, but they are also related to increased in ammatory response and damage 45,46 .Dimensionality reduction t-SNE analysis followed by FlowSOM clustering analysis was performed using Cytobank in order to characterize the lymphoid populations in the lungs of mice.Diverse cell clusters were generated in each condition.Clusters of CD8 + and CD4 + T cells along with clusters of NK cells were increased in the lungs of infected mice, especially in the lungs of non-treated mice (Fig. 6A).Traditional ow cytometry gating using FlowJo showed signi cantly increased numbers of T cells expressing GATA3 (anti-in ammatory T cells -Th2) (Fig. 6B) and T cells expressing FOXP3 (regulatory T cells) (Fig. 6C) in the lungs of treated mice compared to untreated mice.

AT-01-KG protection is FPR2/3-dependent
Lipoxin A 4 binds to FPR2 (FPR2/3 in mice), a member of the formyl peptide receptors (FPRs), triggering and an anti-in ammatory and pro-resolutive response 47 .In order to evaluate if the protection showed by the AT-01-KG treatment was via a direct effect, we infected FPR2/3 -/-mice (KO) with IAV and groups were assigned as mentioned before.We showed that KO treated mice had greater weight lost compared to the WT treated mice and no difference was seen when compared to KO vehicle group.(Fig. 7A).In addition, decreased cell in ltration induced by AT-01-KG treatment was no longer observed in the BALF of KO treated mice (Figs.7B-D).Interestingly, the absence of FPR2/3 increased mononuclear cell in ltration (Fig. 7C) and tissue in ammation (Figs.7E-G) as showed by the in ammatory histopathologic score (Fig. 7H).

DISCUSSION
Lipoxin A 4 is one of the four forms of lipoxins rst described in the 80's 48 .It is able to improve in ammatory lung diseases 49,50 and has an important role in tissue regeneration and homeostasis 51 .
The activation of the lipoxin pathway blocks the production of reactive oxygen species (ROS) 52 , which are associated with tissue damage in IAV infection 11 .Moreover, in 2020, Wang et al. showed that in a model of ventilator induced-lung injury, rats treated with LXA 4 presented improved capillary permeability and reduction in tissue damage 53 .Our results align with those mentioned above since we showed that treatment with LXA 4 reduced pulmonary damage caused by IAV infection.A therapeutic candidate able to decrease lung damage during this infection is highly relevant due to the fact that patients with IAVinduced acute respiratory failure require ventilatory support and the mechanical ventilation itself exacerbate the pulmonary damage impairing the survival rate 54 .
Tissue damage and impaired survival rate are caused by excessive cell recruitment leading to a persistent ongoing in ammation 55 .Recruited cells are responsible for the production of proin ammatory cytokines important for virus clearance, which leads to infection and in ammation control 56,57 .However, lung in ammatory responses can be a double-edged sword since it is associated with greater tissue damage and mortality 58 .In that regard, the production of anti-in ammatory and proresolving mediators is crucial to counter-balance the pro-in ammatory environment induced by the infection 59,60 .Here, the treatment with LXA 4 reduced cell recruitment to the BAL of treated mice consistent with other studies showing that LXA A blocks in ammatory cell migration 61,62 .
The reduction of cell recruitment along with activation of neutrophil apoptosis and their efferocytosis by alternatively activated macrophages are crucial steps to switch on the resolution phase and prepare the tissue to return to homeostasis 52,63 .It has been demonstrated that LXA 4 is able to enhance the resolution of in ammation through the activation of apoptotic pathways in neutrophils 64 but inhibiting apoptosis in macrophages allowing the clearance of apoptotic cells in the tissue 65 .Efferocytosis is the clearance of apoptotic cells by macrophages which is followed by macrophage reprogramming towards an alternatively activated stage 66 when super cial and internal anti-in ammatory cell makers such as CD206 and Arg1 are overexpressed 67,68 .Some studies have demonstrated the association between increased survival rate of in uenza infected mice and decreased numbers of classically activated macrophages, together with the increased expression of alternatively activated macrophages markers 69,70 .Here we demonstrated that treatment with LXA 4 reduced the frequency of classically activated exudate and interstitial macrophages.Treatment also increased the frequency of alternatively activated interstitial macrophages.Altogether, our in vitro and in vivo data suggests that the ability of LXA 4 to activate apoptosis and efferocytosis and to induce macrophage reprogramming appears to contribute to the better outcome observed in this model of IAV infection.
Severe cases of IAV infection are marked by exuberant cytokine production with persistent innate immune cell in ltration and an exuberant T cell response 71 .Serum samples of patients with in uenzaassociated pneumonia showed a positive correlation between poor prognosis and elevated levels of IL-6, IFN-γ and G-CSF 72 .In mice, the production of those cytokines was also correlated with persistent neutrophil survival and in ltration, along with increased tissue damage 44,73 .Here, we also con rmed that correlation, which was improved by the treatment with LXA 4 .We also showed that the treatment increased the frequency of anti-in ammatory T cells, Th2, and regulatory T cells, T regs .In fact, the modulation of the adaptive immune response towards those subsets is associated with inhibition of neutrophil recruitment and better survival 74 .In addition, LXA 4 induced increased frequency of macrophages which can be explained by higher levels of IL-12p40 in the lungs of treated mice since IL-12p40 induces macrophage in ltration 75,76 .
The anti-in ammatory and pro-resolutive effect of both LXA 4 and FPR2 have been investigated not only in IAV infection, but also in other models of infection.Zhu, X. et al showed inhibition of pro-in ammatory mediators by LXA 4 treatment in an Aspergillus fumigatus keratitis mouse model and that antiin ammatory effect was reversed by the blockage of FPR2 77 .In 2017, another study showed that mice co-infected with IAV and Streptococcus pneumoniae presented an increase in neutrophil recruitment and neutrophil activation along with increased levels of proteins in BALF.Co-infected mice were then treated with resolving D1 -aspirin-triggered resolvin D1 (At-RvD1), another pro-resolving mediator that binds to FPR2.The treatment reduced lung damage and elastase activity as well as recruited neutrophils and levels of myeloperoxidase.Interestingly, FPR2 expression was elevated in the lungs of those mice 78 .Additionally, Schloer et al. showed the protective effect of the annexin A1/FPR2 pathway in a model of IAV infection.They showed that mice previously treated with annexin A1 and infected with IAV PR8 presented increased survival and decreased levels of IL-1β, IL-6 e MCP-1 on the third day post infection if compared with non-treated mice.The treatment also decreased lung damage and increased alveolar macrophages through FPR2 activation 79 .Here, our results indicate that, indeed, LXA 4 /FPR2 pathway has a protective role in a model of IAV infection since the protection induced by the treatment was reversed by the absence of FPR2/3.It is of interest to note that in FPR2/3-/-mice greater damage is observed compared to wild type mice.These observations are analogous to exacerbations of disease in models of arthritis and polymicrobial sepsis in FPR2/3 −/− mice highlighting the critical role of this receptor in immune homeostasis 80,81 .

CONCLUSION
Altogether, our results suggest that treatment with a LXA 4 analogue post-IAV infection has a promising therapeutic effect.The effects of the compound are FPR2-dependent and work by inducing resolution of pulmonary in ammation, generating a regulatory phenotype in T cells and macrophages and decreasing tissue damage and mortality.

Figure 2 AT
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Figure 3 A
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Figure 4 AT
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Figure 7 AT
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