**Targeting AnxA1**/**Formyl Peptide Receptor 2 Pathway A**ff**ords Protection against Pathological Thrombo-Inflammation**

### **Shantel A. Vital 1, Elena Y. Senchenkova 1, Junaid Ansari 1,2 and Felicity N. E. Gavins 1,2,3,\***


Received: 25 September 2020; Accepted: 4 November 2020; Published: 13 November 2020

**Abstract:** Stroke is a leading cause of death and disability globally and is associated with a number of co-morbidities including sepsis and sickle cell disease (SCD). Despite thrombo-inflammation underlying these co-morbidities, its pathogenesis remains complicated and drug discovery programs aimed at reducing and resolving the detrimental effects remain a major therapeutic challenge. The objective of this study was to assess whether the anti-inflammatory pro-resolving protein Annexin A1 (AnxA1) was able to reduce inflammation-induced thrombosis and suppress platelet activation and thrombus formation in the cerebral microvasculature. Using two distinct models of pathological thrombo-inflammation (lipopolysaccharide (LPS) and sickle transgenic mice (STM)), thrombosis was induced in the murine brain using photoactivation (light/dye) coupled with intravital microscopy. The heightened inflammation-induced microvascular thrombosis present in these two distinct thrombo-inflammatory models was inhibited significantly by the administration of AnxA1 mimetic peptide AnxA1Ac2-26 (an effect more pronounced in the SCD model vs. the endotoxin model) and mediated by the key resolution receptor, Fpr2/ALX. Furthermore, AnxA1Ac2-26 treatment was able to hamper platelet aggregation by reducing platelet stimulation and aggregation (by moderating αIIbβ<sup>3</sup> and P-selectin). These findings suggest that targeting the AnxA1/Fpr2/ALX pathway represents an attractive novel treatment strategy for resolving thrombo-inflammation, counteracting e.g., stroke in high-risk patient cohorts.

**Keywords:** thrombosis; inflammation; Annexin A1; formyl peptide receptors; sickle cell disease; sepsis

### **1. Introduction**

Stroke is a leading cause of death and disability, with functional impairments producing significant losses in quality of life and accompanying financial burden [1–3]. Although the exact mechanisms responsible for post-ischaemic cerebral damage in stroke remain undefined, the intertwined processes of thrombosis and inflammation play crucial roles in the pathophysiology [4–6]. Unregulated thrombo-inflammation, which involves a complex relationship between inflammatory leukocytes (e.g., neutrophils), platelets, and the vascular endothelium, is also associated with a number of comorbidities such as sickle cell disease ("SCD" [7,8]) and infections (e.g., sepsis [9,10]), which predispose individuals to ischaemic stroke. In the case of SCD, ~11% of SCD patients have a stroke before the age of 20, increasing to 24% by the age of 45 [11]. Furthermore, stroke patients

are not only more susceptible to bacterial infections, but infection itself is an independent risk factor for stroke and a major contributor to worse outcome post stroke, increasing recurrent stroke risk [4]. Therefore, reducing and resolving the impact and detrimental effects of microvascular thrombosis and inflammation associated with underlying co-morbidities, such as those already discussed, represent a major therapeutic challenge.

It is now widely accepted that endogenous pro-resolving mediators released during an inflammatory response play a critical role in effective recovery from inflammation and repair [12,13]. Resolution of inflammation is a tightly orchestrated process, involving specific endogenous mediators such as Annexin A1 ("AnxA1") and its biologically active N-terminal domain, "Ac2-26" (Ac-AMVSEFLKQAWFIENEEQEYVQTVK); Lipoxins e.g., "Lipoxin A4" (5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid) and aspirin-triggered lipoxin A4 (15(R)-epi-LXA4, "ATL"); resolvins ("Rv") e.g., "RvD1" and "RvD2"; protectins e.g., protectin D1 (10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid); maresins e.g., maresin 1 (7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic, "MaR1") [12,14–16]. These specific endogenous mediators moderate and resolve inflammation through protective pro-resolution pathways such as the formyl peptide receptor 2 (also termed "FPR2/ALX", Fpr2, or ALX receptor i.e., the receptor for LXA4) pathway, which is a key resolution pathway [17–22].

The human FPR family consists of three specific receptors termed "FPR1", FPR2/ALX, and "FPR3", all of which are well conserved G-protein-coupled receptors expressed in a host of different cells and tissues, all having pluripotent and diverse roles in the initiation, propagation, and resolution of inflammation [17,19]. All three receptors are clustered together on chromosome 19q13.3 and share significant sequence homology, with FPR1 sharing ~69% and 56% sequence homology with FPR2/ALX and FPR3, respectively, and FPR2/ALX sharing 83% sequence homology with FPR3 [23].

AnxA1 is a 37 kDa protein that belongs to the annexin superfamily of Ca2+-dependent phospholipid binding proteins. It is an anti-inflammatory, pro-resolving protein that is up-regulated by glucocorticoids. Annexins share a common structure formed of a core region and a unique N-terminal domain, which acts as the fingerprint for each annexin of the superfamily. A number of investigations have focussed on the anti-inflammatory effects of AnxA1 and its mimetic peptides (including the blockade of leukocyte recruitment, inhibition of cytokine release, promotion of apoptosis, stimulation of phagocytosis, and decreasing vascular permeability) [17] in a variety of clinically related disease models [17,20,24–28], often via an engagement with FPR1 or FPR2/ALX (murine orthologues Fpr1 and Fpr2/ALX, respectively).

In the context of an ischaemic stroke setting, we have previously shown that AnxA1 (via Fpr2/ ALX) is able to act both as a therapeutic and a prophylactic drug, reducing infarct volume and improving stroke outcome in a mouse model of acute experimental stroke [17,29] without increasing the risk of intracerebral haemorrhage [3,29,30]. Moreover, we demonstrated that this anti-inflammatory/pro-resolving compound was able to act as an anti-thrombotic agent suppressing thrombin-induced inside-out signalling events such as Akt activation, intracellular calcium release, and Ras-associated protein 1 ("Rap1") [31], thereby exerting protection by altering platelet phenotypes from pro-pathogenic to regulatory [29]. These findings provided novel physio-pharmacological properties of AnxA1 as a powerful pro-resolving mediator of thrombo-inflammation and opened new avenues for an attractive therapeutic treatment strategy for patients with stroke. However, the anti-thrombotic effect of AnxA1 in patients with co-morbidities susceptible to stroke is less well characterized. Therefore, the objective of this current study was to use pharmacological and genetic approaches together with a photoactivation thrombosis (light/dye) model to test the hypothesis that targeting the AnxA1/FPR-pathway protects the cerebral microvascular system against inflammation-induced microvascular thrombosis in co-morbidities susceptible to stroke.

### **2. Materials and Methods**

### *2.1. Animals*

All studies were done blinded and performed on adult male mice weighing 25–30 g. C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The sickle cell transgenic mice (STM) (also termed β<sup>s</sup> mice, heterozygous BERK, trait BERK, or sickle cell trait model) were homozygous for knockout of murine α-globin and heterozygous for knockout of murine β-globin and had one copy of the linked transgenes for human α- and βs-globins [32]. STM used in this study were a gift from Professor Robert Hebbel (University of Minnesota). STM were bred onsite and showed no obvious phenotype and were fertile. Mice were maintained on a 12 h (h) light–dark cycle, during which room temperature was maintained at 21–23 ◦C. Mice had access to a standard chow pellet diet and tap water *ad libitum*. All animal experiments were approved by the Louisiana State University Health Sciences Center—Shreveport (LSUHSC-S) Institutional Animal Care and Use Committee (IACUC), were in accordance with the guidelines of the American Physiological Society and complied with ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

### *2.2. Endotoxin (LPS) Administration*

Mice were injected intraperitoneally (i.p.) with "LPS" (Escherichia coli serotype 0111:B4, purified initially by phenol extraction and purified further by ion exchange chromatography. L3024. Sigma-Aldrich, St Louis, MO, USA), two hours before the experiment at a dose of 0.4 mg/kg in 100 μL vehicle sterile saline [33].

### *2.3. Photoactivation Thrombosis Model. (Light*/*Dye Method)*

*Intravital microscopy ("IVM")* was performed as previously described [29]. Briefly, mice were anaesthetized via i.p. injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg), and the femoral vein was cannulated for dye (fluorescein isothiocyanate (FITC)-dextran) administration. The head of each mouse was fixed in a frame (sphinx position) and the parietal bone was exposed by a midline skin incision, followed by a craniectomy [29]. A total of 10 mg/kg of 5% FITC dextran (150,000 mol wt, Sigma-Aldrich) was injected i.v. and allowed to circulate for ten minutes (mins) prior to photoactivation. Photoactivation was initiated (excitation, 495 nm; emission, 519 nm) by exposing 100 μm of vessel length to epi-illumination with a 175-W xenon lamp (Lambda LS, Sutter, Novato, CA, USA) and a fluorescein filter cube (HQ-FITC, Chroma, San Francisco, CA, USA) [29]. The excitation power density was measured daily (ILT 1700 Radiometer, SED033 detector; International Light, Peabody, MA, USA) and maintained within 1% of 0.77 W/cm2. Epi-illumination was applied continuously, and onset and blood flow cessation times were quantified in cerebral vessels (diameter range: 30–70 μm) until blood flow had ceased in the vessel under study [29]. In some cases, 20 min prior to onset of thrombosis, mice were treated with vehicle (saline), AnxA1Ac2-26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK, 4 mg/kg. Cambridge Research Biochemicals, Cambridge, UK), pan FPR antagonist Boc2 (N-tert-butoxycarbonyl-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe, 0.4 mg/kg; MP Biomedicals, Cambridge, UK) or FPR2/ALX antagonist WRW4 (2.2 mg/kg. Tocris, Bristol, UK) administered (100 μL) i.v. [20,29]. Doses/concentrations used in the study are congruent with binding affinities for the Fprs and were chosen based on our previous findings [17,20,29]. All compounds were made in vehicle sterile saline.

### *2.4. Bleeding Time*

Bleeding times were quantified in mice treated with vehicle saline, LPS, or AnxA1Ac2-26, as previously described. Briefly, mice were anaesthetized with i.p. injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg). A small tail segment (3 mm) was cut cleanly with a scalpel blade, and bleeding was monitored at 15-s intervals by absorbing the bead of blood with filter paper without contacting

the wound site [29]. When no blood was observed on the paper, bleeding was determined to have ceased [34].

### *2.5. Platelet Cell Counts*

Platelets from peripheral blood were stained with 1% buffered ammonium oxalate and counted using a haemocytometer.

### *2.6. Platelet Flow Cytometry*

Whole blood was collected (0.9 mL) via carotid artery into a syringe containing 0.1 mL anticoagulant citrate dextrose ("ACD") buffer, transferred into an Eppendorf tube, and centrifuged at 1200 rpm for 8 min. The platelet-rich plasma ("PRP") layer was transferred to a new Eppendorf tube and centrifuged at 3000 rpm for 10 min. The supernatant was removed and the pellet was resuspended in Tyrodes buffer (containing 1 mM Ca2+) along with the appropriate antibody and allowed to incubate for 15 min at 37 ◦C. The reaction was stopped by the addition of 450 μL of 1% paraformaldehyde. P-selectin and JON/A-PE FITC antibodies (Emfret Analytics, Eibelstadt, Germany) were used to measure platelet activation and surface P-selectin exposure in murine platelets using flow cytometry, as previously described [35,36]. IgG isotype antibodies were used as controls. Briefly, the platelets were treated with Fc block (15 min, room temperature) to inhibit non-specific binding according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). This was followed by treatment with AnxA1Ac2-26 (30 μM) or vehicle for 30 min and then, the addition of antibodies (1:8 dilution). Next, the glycoprotein VI (GPVI) collagen receptor agonist convulxin (CVX) (1.7 ng/mL. Cayman Chemical Company, MI, USA) was used to stimulate the platelets for 15 min. In some cases, prior to CVX, platelets were treated with LPS (7.5 μg/mL). The activation was stopped by the addition of 450 μL of 1% paraformaldehyde. Platelets were identified by their light scattering using an LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and Diva8 software by assessing at least 10,000 events per sample, as previously described [34].

### *2.7. Platelet Aggregation Assay*

Arterial blood was freshly collected from the carotid artery of mice, as described above. Platelets 8–10 <sup>×</sup> 106/mL were used to monitor platelet aggregation velocity after agonist exposure in a cuvette loaded with 6 mL of platelet media (140 mM NaCl, 10 mM HEPES, 10 mM NaHCO3, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM D-glucose, pH 7.4) using a laser particle analyser (LasCa-1C, Lumex Ltd., St. Petersburg, Russia) by a low angle light scattering method, as previously described [29]. Platelets were administered either vehicle (saline) or LPS (7.5 μg/mL) and the normalized velocity of aggregation was calculated using original software LasCa\_32, as previously described [29].

### *2.8. Statistical Analysis*

All data were analysed using GraphPad Prism8 software. Data are shown as mean values ± standard error of the mean (SEM), with *n* values given in the respective figures. Results from thrombosis experiments were confirmed to follow a normal distribution using a Kolmogorov–Smirnov test of normality with Dallal–Wilkinson–Lillie for the corrected *p* value. Data that passed the normality assumption were analysed using Student's *t*-test (two groups) or ANOVA with Bonferroni post hoc tests (more than two groups). Data that failed the normality assumption were analysed using the non-parametric Mann–Whitney U test (two groups) or Kruskal–Wallis with Dunn's test (more than two groups). Differences were considered statistically significant at a value of *p* < 0.05.

### **3. Results**

### *3.1. AnxA1Ac2-26 Inhibits LPS-Induced Thrombus Formation in Cerebral Microcirculation of C57*/*BL6 Mice*

The light/dye model coupled with intravital microscopy was used to quantify thrombus formation in cerebral arterioles and venules, as determined by onset time (i.e., the time to onset of visible aggregates) and blood flow cessation time (Figure 1A). Figure 1B,C shows that onset time was significantly faster in both arterioles and venules in LPS-treated mice vs. saline (vehicle)-treated mice (with the exception of LPS+AnxA1Ac2-26+Boc2). We found that administration of AnxA1Ac2-26 delayed the time of onset in the venules of mice treated with LPS vs. LPS alone (*p* < 0.05), suggesting a propensity for the AnxA1 peptide to initiate an early anti-thrombo-inflammatory response against inflammation-induced thrombosis.

**Figure 1.** AnxA1Ac2-26 protects against the effects of endotoxin (LPS) in light/dye-induced thrombosis responses in the cerebral microcirculation. (**A**) Schematic showing the experimental protocol: (i) Mice (C57BL/6) were subjected to vehicle (saline) or LPS (0.4 mg/kg) for 2 h. (ii) Mice were treated with vehicle (saline) or compound(s): AnxA1Ac2-26 (4 mg/kg) with/without pan FPR antagonist Boc2 (0.4 mg/kg) or FPR2/ALX antagonist WRW4 (2.2 mg/kg) 20 min prior to thrombosis. (iii) A cranial window was performed and (iv) fluorescein isothiocyanate (FITC)-dextran was injected (10 mg/kg of 5%). (v) Mice were subjected to intravital microscopy and light/dye-induced thrombus formation, with onset and blood flow cessation times recorded for cerebral (**B**) arterioles and (**C**) venules. Data are means ± SEM of 5–6 mice/group. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 vs. corresponding vehicle (saline). # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. corresponding LPS group. \$\$\$\$ *p* < 0.0001 vs. same group for onset time. ˆ *p* < 0.05, ˆˆˆ *p* < 0.001 vs. corresponding LPS+AnxA1Ac2-26 group.

As previously reported in C57BL/6 mice [34], LPS treatment augmented thrombus formation in the cerebral microcirculation (as noted by the decreased blood flow cessation times in LPS-treated mice vs. their saline-treated counterparts). This pro-thrombotic environment was mitigated by the treatment of AnxA1Ac2-26, which was effective in both vessel types analysed (arterioles: 16.7 ± 1.0 min vs. 30.5 ± 2.7 min; venules: 9.3 ± 1.3 min vs. 17.3 ± 2.4 min; LPS vs. LPS+ AnxA1Ac2-26, respectively).

Having established the anti-thrombotic effect of AnxA1Ac2-26 against inflammation-induced thrombosis, we next ascertained whether these effects were mediated by the classic AnxA1/Fpr pathway. Figure 1B,C shows the FPR pan-antagonist Boc2 inhibited the actions of the AnxA1 mimetic peptide (*p* < 0.0001) in cerebral arterioles, but not in venules, suggesting a vessel-specific mechanism of action via the FPR family. (No effect was observed in either onset or blood flow cessation times for Boc2 administration alone vs. LPS; see Figure S1).

To further expand on these findings and tease out through which member(s) of the FPR family AnxA1Ac2-26 was exerting its protective effects, mice were co-administered AnxA1Ac2-26 with WRW4 (specific FPR2/ALX antagonist). We found that WRW4 blocked AnxA1Ac2-26 afforded protection in both cerebral arterioles (*p* < 0.001) and venules (*p* < 0.05), thereby confirming an FPR2/ALX mechanism (Figure 1B,C). (Figure S1 shows that no effect was noted in either onset or blood flow cessation times for administration of WRW4 alone vs. LPS). These data further support the growing body of evidence that the FPR2/ALX pathway plays a crucial role in both thrombotic and inflammatory pathways in the brain microvasculature.

### *3.2. AnxA1Ac2-26 Reduces the E*ff*ect of Endotoxin-Induced Platelet Activation*

Since AnxA1Ac2-26 was able to reduce thrombosis, we next sought to determine whether exogenous administration of AnxA1Ac2-26 had any effect on the haemostatic activity of platelets. Figure 2A shows that AnxA1Ac2-26-treated LPS mice exhibited decreased bleeding times (*p* < 0.001), and these observations were not related to changes in circulating platelet cell counts as AnxA1Ac2-26 administration had no effect on the LPS-heightened platelet cell counts (Figure 2B). The interaction of platelets with neutrophils is involved in many thrombo-inflammatory responses. Furthermore, LPS is a potential mediator of neutrophil extracellular traps (NETosis) and induces platelet–neutrophil aggregate formation, and neutrophils have been shown to be essential for platelet recruitment in endotoxaemic models [37,38]. Here, we found that circulating neutrophil counts were heightened in those mice challenged with LPS, but these levels were not modified by AnxA1Ac2-26 (Figure S3). We also found that the peptide was able to act as an anti-aggregant in its ability to moderate platelet aggregation induced by LPS (by reducing the velocity of platelet–platelet aggregate formation; see Figure 2C). These data provide further evidence that not only is the AnxA1 mimetic peptide a known anti-inflammatory drug, but it also has potential as an effective anti-platelet drug.

**Figure 2.** AnxA1Ac2-26 treatment inhibits endotoxin-induced activation of the platelet activation. (**A**) tail bleeding times and (**B**) peripheral blood platelet counts were assessed following saline (vehicle) or AnxA1Ac2-26 (4 mg/kg) administration for 20 min following 2 h saline (vehicle) or LPS (0.4 mg/kg) administration. (**C**) Isolated platelets were treated with saline or LPS (7.5 μg/mL) with or without AnxA1Ac2-26 (1 μg/mL) and the velocity of aggregate formation was measured using a low-angle light-scattering technique. Data are means ± SEM of 5–6 mice/group. *\* p* < 0.05, *\*\*\* p* < 0.001, *\*\*\*\* p* < 0.0001 vs. corresponding vehicle (saline). # *p* < 0.05, #### *p* < 0.0001 vs. corresponding LPS group. ns—non-significant vs. corresponding saline group.

### *3.3. AnxA1Ac2-26 A*ff*ords Protection against Cerebral Thrombo-Inflammation*

Having assessed the anti-thrombotic effect of AnxA1Ac2-26 on endotoxin (LPS)-enhanced thrombosis, a model which is known to favour venular thrombosis [39], we next wanted to determine whether the protective effect of the peptide was model-specific. SCD assumes a pro-inflammatory and pro-thrombotic phenotype throughout the microvasculature. As such, we performed the light/dye thrombosis model in STM, which are known to share these clinical features [32,40,41]. Figure 3 shows that without stimulation, STM assume a pro-thrombotic phenotype, as evidenced by much quicker (~50% quicker) blood flow cessation times than non-STM (Figure S2) in both sides of the vascular tree (arterioles: 33.2 ± 1.8 min vs. 15.1 ± 1.2 min (*p* < 0.05); venules: 15.1 ± 1.2 vs. 6.5 ± 0.4 (*p* < 0.05); non-STM vs. STM). Moreover, in comparison to saline-treated STM, administration of AnxA1Ac2-26 significantly protected (*p* < 0.01) against thrombotic events by causing a significant increase in blood flow in arterioles by 47.8% and in venules by 63.5%, respectively, an effect that was again mitigated by the co-administration of either Boc2 or WRW4 (Figure 3B,C). (No differences were observed in blood flow onset times when either of the two antagonists were administered alone; see Figure S4). Interestingly, AnxA1Ac2-26 also delayed the onset time in venules (Figure 3B), an effect that concurred with the effect on cerebral venules following LPS-induced thrombosis (Figure S2).

### *3.4. AnxA1Ac2-26 Primes Platelet Activation via GPVI Pathway Regulation in SCD-Associated Thrombo-Inflammation*

Having found that AnxA1Ac2-26 was able to modify the thrombo-inflammatory environment by prolonging blood flow cessation in the cerebral microcirculation in both arterioles and venules, we next sought to investigate the influence of this AnxA1 mimetic peptide on a key platelet receptor which is known to play a central role in thrombosis, i.e., GPVI. This receptor is able to stimulate platelet adhesion by its ability to enhance the affinity of other integrins such as αIIbβ<sup>3</sup> via inside-out signalling mechanisms [42]. Figure 3D,E show that stimulation of platelets with the GPVI collagen receptor agonist CVX potentiated both αIIbβ<sup>3</sup> (Figure 3C) and P-selectin in STM (Figure 3D). Direct treatment of platelets with AnxA1Ac2-26 (30 μM) was found to suppress these levels (*p* < 0.05); although the effects on P-selectin expression were significant, they were less dramatic as those observed with αIIbβ3. Taken together, these findings suggest that AnxA1Ac2-26 is able to reduce the susceptibility of platelets to interact with collagen receptors, thereby reducing the propensity for platelets to aggregate and cause thrombosis in a thrombo-inflammatory environment.

### *3.5. Exploiting the AnxA1*/*FPR2*/*ALX Pathway as a Therapeutic Strategy to Alleviate Thrombo-Inflammation*

A pro-inflammatory disease state exists in SCD, which predisposes patients to vaso-occlusion (VOC) in response to triggering factors such as infection [43], thus heightening the risk for stroke [11], acute chest syndrome [44], and early death [45]. As such, in the final part of the study, we validated the therapeutic potential of AnxA1Ac2-26 as a treatment therapy against thrombo-inflammation in STM following LPS injection and photoactivation to induce a VOC (Figure 4A). Markedly, STM mice treated with LPS did not display an additive thrombo-inflammatory response (Figure 4B,C) above that observed in the absence of LPS (Figure 3B,C), suggesting an already exhaustive SCD phenotype. Nonetheless, Figure 4B shows that STM mice treated with AnxA1Ac2-26 caused a prolongation of blood flow cessation responses in arterioles, which was abrogated in the presence of Boc2. This protective effect afforded by the peptide was not mirrored in cerebral venules, despite there being a trend towards an increase (Figure 4C).

**Figure 3.** AnxA1Ac2-26 affords protection against cerebral thrombo-inflammation in STM and modifies cell surface expression of platelet receptors. (**A**) Schematic showing the experimental protocol: (i) sickle cell transgenic mice (STM) were treated with vehicle (saline) or compound(s): AnxA1Ac2-26 (4 mg/kg) with/without pan FPR antagonist Boc2 (0.4 mg/kg) or FPR2/ALX antagonist WRW4 (2.2 mg/kg) 20 min (mins) prior to thrombosis. (ii) A cranial window was performed and (iii) FITC-dextran was injected (10 mg/kg of 5%) and allowed to circulate for 10 min before STMs were subjected to (iv) intravital microscopy and light/dye-induced thrombus formation. Onset time and blood flow cessation times were recorded for cerebral. (**B**) arterioles and (**C**) venules. (**D**+**E**) Activated integrin αIIbβ<sup>3</sup> and P-selectin were quantified using flow cytometry in platelets isolated from STM following 15 min stimulation with GPVI collagen receptor agonist, convulxin (CVX, 1.7 ng/mL). Vehicle (saline) or AnxA1Ac2-26 (30 μM) was given 30 min prior to activation, followed by vehicle (saline) or LPS (7.5 μg/mL for 5 min). (**D**) % of activated integrin αIIbβ<sup>3</sup> (JON-A) and (**E**) % of activated P-selectin expression (CD62P). Data are means ± SEM of 5–6 mice/group. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. corresponding vehicle (saline). @@ *p* < 0.01 vs. corresponding Ac2-26 group. \$\$\$ *p* < 0.001, \$\$\$\$ *p* < 0.0001 vs. same group for onset time. # *p* < 0.05 vs. CVX+vehicle group.

**Figure 4.** AnxA1Ac2-26 affords arteriolar protection against cerebral inflammation-induced thrombosis in sickle cell transgenic mice (STM). (**A**) schematic showing the experimental protocol: (i) STM were subjected to vehicle (saline) or LPS (0.4 mg/kg) for 2 h and (ii) treated with vehicle (saline) or compound(s): AnxA1Ac2-26 (4 mg/kg) with/without pan FPR antagonist Boc2 (0.4 mg/kg) 20 min prior to thrombosis. (iii) A cranial window was performed and (iv) FITC-dextran was injected (10 mg/kg of 5%). (v) Mice were subjected to intravital microscopy and light/dye-induced thrombus formation, with onset and blood flow cessation times recorded for cerebral (**B**) arterioles and (**C**) venules. Data are means <sup>±</sup> SEM of 5–6 mice/group. # *p* < 0.05 vs. LPS group. ˆ *p* < 0.05, vs. LPS+AnxA1Ac2-26 group. ns—non-significant vs. corresponding LPS group.

### **4. Discussion**

The interplay between thrombosis and inflammation (thrombo-inflammation) occurs in a broad range of human disorders (including stroke, sepsis, and SCD), with ensuing complications transpiring to be more hazardous in the microvasculature of injured tissues and organs [46]. Given the devastating impact of pathological thrombo-inflammation, there is an unmet clinical need to understand the complex pathophysiology for therapeutic development of drugs that are more efficacious, have fewer side-effects, and are devoid of bleeding complications that would ultimately undermine the clinical benefit. Here, using pharmacological and genetic approaches together with a photoactivation thrombosis (light/dye) model coupled with intravital microscopy, we provide further evidence of the multifaceted role of AnxA1 N-terminal mimetic peptide AnxA1Ac2-26 as an anti-thrombotic and anti-coagulant agent. Our data demonstrate that AnxA1Ac2-26 affected not only the haemostatic action of platelets (e.g., reduced bleeding times), but moderated platelet aggregation (e.g., diminished the propensity for platelets to form platelet–platelet aggregates) and lowered thrombogenesis (i.e., reduced the time of onset of platelet deposition/aggregation within cerebral vessels), thereby reducing LPS-induced thrombosis. Furthermore, the AnxA1 peptide was able to decrease αIIbβ<sup>3</sup> activation and reduce P-selectin expression elicited by GPVI, inhibiting platelet activation and thrombosis in STM. Taken together, our data reveal that AnxA1Ac2-26 affords protection by altering the haemostatic action of platelets, modulating platelet cell surface molecules (e.g., αIIbβ<sup>3</sup> and P-selectin), and reducing platelet heterotypic aggregation. In so doing, AnxA1Ac2-26 reduces platelet activation, adhesion, and aggregation and tempers thrombosis. Collectively, these results highlight the potential for AnxA1Ac2-26 as a viable therapy for the management of thrombo-inflammatory disorders (as shown in Figure 5).

The resolution of inflammation is a tightly orchestrated process that is controlled by endogenous biosynthetic mediators such as AnxA1 and its mimetic peptide (AnxA1Ac2-26). Both these compounds have been shown to act at various points in the inflammation-resolution pathway [47]. The known anti-inflammatory actions of AnxA1Ac2-26 (e.g., inhibiting neutrophil recruitment, decreasing neutrophil endothelium interactions, and suppressing inflammatory cytokine production [47]) have been widely studied and these actions eventually contribute to inflammation resolution by enabling apoptosis [14] and phagocytosis [29]. Previously, it was thought that inflammation and thrombosis were two independent processes. However, the innate and coagulation systems are so intertwined that these two processes are now considered to be in part the same, in that microvascular thrombosis is accompanied by inflammation and vice versa, an association referred to as thrombo-inflammation [17]. However, a greater understanding of the links between inflammation and thrombosis is needed in order to develop new therapeutic opportunities [29]. Our laboratory is actively pursuing therapeutic drug discovery programs focused on the concept of thrombo-inflammation resolution, with the AnxA1/FPR2/ALX pathway being of significance.

It is estimated that >80% of sepsis patients have either clinical or subclinical hypercoagulopathy, increasing the risks for both thrombosis and haemorrhage [48,49]. These increased thrombotic risks can function not only as acute triggers for stroke but are associated with poor long-term prognosis after stroke [48]. Endotoxin (LPS) is a component of Gram-negative bacteria cell walls, producing many clinical manifestations of Gram-negative sepsis [50]. Using an animal model of endotoxaemia, we found AnxA1Ac2-26 treatment counteracted the enhanced microvascular thrombus formation, an effect which was found to be mediated through Fpr2/ALX. These protective effects are in line with the defensive actions of AnxA1Ac2-26 on the inflammatory cascade that have been observed previously in the microcirculation post-LPS challenge. In these studies, AnxA1Ac2-26 was able to abrogate leukocyte adhesion and plasma protein extravasation in the brain [33] and the mesentery. Furthermore, AnxA1 and AnxA1Ac2-26 both cause leukocyte detachment, reducing the inflammatory environment and driving resolution [51]. Within this study, we have found that although AnxA1Ac2-26 treatment prolonged blood flow cessation time within a thrombotic LPS environment, it also lowered thrombogenesis (i.e., reduced the time of onset of platelet deposition/aggregation within cerebral vessels). Previously, we have shown that whole protein AnxA1 treatment reduces platelet adhesion to the cerebral endothelium following cerebral I/RI [17]. However, although thrombosis and inflammation are interlinked processes, the lack of effect on platelet adhesion observed here may relate to the type of model (thrombotic vs. inflammatory) and/or the possibility that, as with the parent compound in cerebral I/RI, AnxA1Ac2-26 is orchestrating a complex change in the platelet phenotype from pro-pathogenic to regulatory (which concurs in cerebral I/RI [29]) and in doing so, enhances blood flow cessation times.

In the clinic, the pro-thrombogenic phenotype observed in sepsis patients is often accompanied by thrombocytopenia (with prolonged time span of thrombocytopenia being correlated with increased mortality in intensive care patients [52]), reactivity of platelets and by an imbalance between procoagulant and anticoagulant mechanisms [49]. Thus, platelet count is a useful marker of adverse disease activity, although it remains unclear as to whether the reduction in the number of platelets is mechanistically linked to heightened platelet activity and increased thrombosis. Here, LPS treatment caused thrombocytopenia which concurred with previous findings, not only by our group [53], but also by others e.g., George et al. showed LPS to induce severe thrombocytopenia and inflammation resulting in spontaneous intra-alveolar haemorrhage [54] and Hillgruber et al. concluded that thrombocytopenia in the skin and lungs can be limited by targeting neutrophil diapedesis through the endothelial barrier [55], demonstrating the importance of the cross-talk between neutrophils and platelets. In our study, although AnxA1Ac2-26 did not affect thrombocytopenia induced by LPS (which may relate to the mechanisms that regulate thrombosis and haemostasis), it did reduce bleeding time in an in vivo assay that is widely used to assess the haemostatic action of platelets [56]. These findings are of clinical significance because the efficacy of anti-platelet agents is often limited by bleeding complications. Genetic *AnxA1* deletion is not associated with spontaneous thrombosis or bleeding but leads to exacerbated cerebral inflammatory responses following experimental ischaemic stroke [29]. Furthermore, decreased circulating levels of AnxA1 are present in thrombo-inflammatory conditions including ischaemic stroke, SCD, sepsis, Crohn's disease, and obesity [29]. Taken together, these results demonstrate AnxA1Ac2-26 attenuates LPS-induced peripheral bleeding and intravascular thrombosis, affording a therapeutic strategy for thrombotic complications.

Defining molecular mechanisms regulating thrombo-inflammation in specific disease states is of major clinical importance. Having characterised the effects of AnxA1Ac2-26 on inflammation-induced thrombosis instigated by LPS, we next wanted to ascertain whether the protective effects elicited by the AnxA1 peptide were specific to the LPS experimental model of thrombo-inflammation. As such, we focused on SCD, an inherited autosomal recessive disorder (resulting from a single amino acid substitution in the haemoglobin β chain) whose pathophysiology is characterized by relentless thrombo-inflammation, enabling heightened propensity for I/RI events such as stroke [7,8].

As observed with mice stimulated with LPS, STM had comparable cerebral responses in both arterioles and venules when exposed to light/dye thrombosis, supporting findings demonstrating that a pro-inflammatory phenotype exists within their cerebral microvasculature (as quantified by enhanced leukocyte–endothelial cell adhesion and increased ROS production [32,57]). AnxA1Ac2-26 treatment reduced cerebral thrombosis and the use of Fpr antagonists Boc2 and WRW4 confirmed that these protective effects were caused by receptor engagement of peptide AnxA1Ac2-26 to Fpr2/ALX. Furthermore, research from our laboratory (Ansari et al., 2020, under review) has also shown AnxA1Ac2-26 to reduce citrullinated histone-3 (H3Cit+)-rich neutrophil extracellular trap (NET) production in SCD. These findings (along with those from other groups [58,59]) are of clinical significance as NETs have been recognized as critical components for venous and arterial thrombosis and inhibition of pathological NET formation may be beneficial for thrombo-inflammatory events and disorders such as SCD [59]. Taken together, these results demonstrate the versatility and innovative approach of AnxA1Ac2-26 as a therapeutic compound for the resolution of thrombo-inflammation.

We uncovered that although the Fpr-pan antagonist Boc2 abrogated the effects of the peptide in arterioles, no statistically significant effect was observed in venules irrespective of the thrombo-inflammatory environment, although there was a trend towards abrogation. The same could not be said for the Fpr2/ALX-specific antagonist WRW4, which was effective in annulling the peptide's responses irrespective of vessel type or thrombo-inflammatory model. This dichotomy of behaviour of the peptide is less likely to be due to antagonist doses (as these were chosen based on dose–response curves) but could be due the fact that FPRs have a large number of diverse unrelated ligands that are able to bind to the receptor family to elicit pro- and anti-inflammatory effects that are specific to ligand and cell type. More recently, the concept of biased agonism has been coined to describe "the ability of a ligand to selectively activate subsets of downstream signalling pathways coupled to a receptor while inhibiting others" [60], which could help to explain differences observed between AnxA1Ac2-26 and co-administration with either the pan-antagonist Boc2 or the Fpr2-specific antagonist WRW4. Other factors that could be accountable for the variations in peptide and antagonist responses could be the known physiological differences between arterioles and venules e.g., shear rates (being predominantly higher in arterioles vs. venules) and differences in the thrombi formed in these respective vessels e.g., arteriolar thrombi being platelet-rich, but venular thrombi also containing leukocytes (typically near the surface of the microvascular thrombi) [61]. Despite these disparities, our data demonstrate the potent activity and versatility of AnxA1Ac2-26 to mitigate both LPS- and

SCD-associated cerebral thrombosis (in both arterioles and venules) in an Fpr2/ALX-dependent manner. Correspondingly, whole protein AnxA1 has also been shown to afford protection against subsequent thrombotic events post stroke, demonstrating the diversity of AnxA1 and its mimetic peptide [29].

Platelets play a pivotal role in normal haemostasis and thrombus formation, and more recently have been found to also play a role in maintaining barrier function [6]. Once activated, platelets undergo a shape change to enhance adhesiveness, a process mediated by exposed glycoprotein receptors on the platelet surface [6]. GPVI is a unique platelet membrane glycoprotein whose binding with collagen (which is exposed on the extracellular matrix following stroke [6]) results in platelet activation and adhesion, and ultimately, thrombus formation. Platelets primarily rely on signalling through GPVI and C-type lectin-like type II transmembrane receptor (CLEC-2) to prevent bleeding [62]. GPVI, via inside-out signalling, enhances the affinity of integrins such as αIIbβ3 (the most abundant platelet receptor (80,000–100,000 copies per platelet) and necessary for aggregation), leading to platelet adhesion [53]. As such, given the important role that GPVI plays in thrombosis, we used flow cytometry to investigate whether the anti-thrombotic effects of AnxA1Ac2-26 involved GPVI signalling. By directly activating the GPVI pathway using CVX, we were able to discern that the AnxA1 peptide was able to decrease αIIbβ<sup>3</sup> activation and the surface expression of P-selectin, which in turn inhibits platelet activation. Interestingly, we have previously found that the parent protein does not reduce P-selectin expression in thrombin-stimulated platelets [29]. One might speculate that these discrepancies may lie in the different platelet stimuli and/or the downstream responses elicited by the binding of either the whole protein or the peptide to Fpr2/ALX and subsequent conformational changes of the receptor [26]. Equally, these effects may simply relate to the varied regulatory functions afforded by the peptide during the host defence response. Further experiments will help to tease out these mechanisms.

Other regulatory mechanisms (such as those involving cyclooxygenase 2 ('COX2')/ hydroxyeicosatetraenoic acid-5 (HETE-5)/Lipooxygenase) may also be involved in the role that the AnxA1/Fpr2/ALX pathway plays in thrombo-inflammation. AnxA1 (and its mimetic peptide AnxA1Ac2-26), Lipoxin A4, and ATL (produced after aspirin acetylation of inducible COX-2) all exert their protective effects through FPR2/ALX at different phases of the inflammatory response. Phospholipase (e.g., calcium-dependent cytosolic phospholipase A2 ("cPLA2")) activity releases arachidonic acid ("AA") from phospholipids in the outer nuclear membrane. Once released, the free fatty acid can be metabolized *via* enzymatic pathways including the (COX) and lipoxygenase (LOX) pathways, generating 2-series prostaglandins (PGs) and thromboxanes (Txs) (COX pathway) or 4-series leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) (LOX pathway) [63]. Transcellular production of lipoxins and leukotrienes ensues when contact is made between e.g., neutrophils and platelets. Previous research from our laboratory has shown that when aspirin is administered to mice with experimental stroke, they can produce ATL, which triggers pro-resolving responses through the engagement of the AnxA1/Fpr2/ALX pathway [17]. Furthermore, ASA administered to healthy volunteers is also capable of producing bioactive levels of ATL [64]. More recently, Sanches et al. showed that macrophages lacking AnxA1 have increased AA metabolism and eicosanoid production. This lack of AnxA1 favours LPS "over-priming" and exacerbated NLR Family Pyrin Domain Containing 3 ("NLRP3") activation, demonstrating an important role for AnxA1 in inflammasome activation [63]. Other studies have also shown AnxA1 to exert its effects via the FPR2/ALX/p38 mitogen-activated protein kinase ("MAPK")/COX-2 pathway in an experimental model of intracerebral haemorrhage, although other MAPKs may also be involved [65].

Patients with thrombo-inflammation (including stroke, sepsis, and SCD) all present with abnormal circulating platelet–leukocyte aggregates (with platelets binding to neutrophils in a P-selectin-dependent mechanism, an interaction that primes the leukocyte and promotes integrin activation), increasing their propensity to develop disseminated intravascular coagulation [49]. Studies have indicated that ischaemic stroke is not simply mediated by platelet aggregation, but also by other intravascular cells including neutrophils, although it remains unclear whether the direct interaction between platelets and neutrophils is critical for the pathogenesis of ischaemic stroke [59]. Von Brühl et al. demonstrated leukocytes (predominantly neutrophils) crawl and adhere to the endothelium, initiating and propagating venous thrombosis [66]. Additionally, neutrophils have now been shown to be involved in arterial thrombosis as well [67], although platelet activation and aggregation is the main driving factor. Furthermore, several distinct mechanisms have been postulated for neutrophil involvement in thrombosis including: the transfer of tissue factor from neutrophils to platelets, inducing thrombosis [68]; the release of specific mediators that affect thrombosis, including cathepsin G and neutrophil elastase (which inactivate anticoagulant systems such as tissue factor pathway inhibitor, thrombomodulin, and antithrombin), neutrophil oxidants (which e.g., inactivate thrombomodulin and ADAMTS13), and micro-RNAs [59]; and the generation of NETs which can trap and activate platelets via histone production [69]. Of interest, more recently, COVID-19 patients have been shown to have increased serum markers of NETs including myeloperoxidase-DNA (MPO-DNA) and H3Cit. Given our recent findings (Ansari et al., 2020, under review) that AnxA1Ac2-26 is able to reduce H3Cit+-rich NET production, transforming neutrophil phenotype from pro-NETotic to pro-apoptotic (thereby driving thrombo-inflammation resolution in SCD), we are currently investigating whether these findings can be exploited to provide therapeutic value for COVID-19 patients [70].

**Figure 5.** Anti-inflammatory and pro-resolving effects of targeting the AnxA1/Fpr2/ALX pathway against thrombo-inflammation. Thrombo-inflammatory conditions such as sepsis [35] and sickle cell disease (SCD) [71] induce inflammation (Box 1) as well as thrombus formation (Box 2) in the cerebral microcirculation enabling thrombo-inflammation (as indicated by e.g., heightened platelet GPVI, αIIbβ<sup>3</sup> and P-selectin expression, and increased platelet–platelet (homotypic) aggregates and platelet-neutrophil (heterotypic) aggregates). Neutrophils also produce neutrophil extracellular traps (NETs) which can exacerbate thrombo-inflammation (Box 3). AnxA1Ac2-26 inhibits lipopolysaccharide (LPS) and SCD-induced cerebral inflammation (Box 4) and cerebral thrombosis (Box 5) via Fpr2/ALX. The AnxA1 peptide affords protection by altering the haemostatic action of platelets, modulating platelet cell surface molecules elicited by GPVI (e.g., αIIbβ<sup>3</sup> and P-selectin), and reducing platelet (homotypic and heterotypic) aggregation. Thus, AnxA1Ac2-26 promotes the resolution of thrombo-inflammation by reducing platelet activation, adhesion, and aggregation which cause and promote thrombosis (Box 6).

### **5. Conclusions**

In conclusion, we provide strong supporting evidence that AnxA1 mimetic peptide AnxA1Ac2-26 possesses an arsenal of immune responses extending beyond that of an anti-inflammatory (e.g., attenuation of leukocyte-platelet responses post stroke, reduction of lipopolysaccharide-induced leukocyte adhesion and migration) and pro-resolution mediator to also include a role as an anti-coagulant and anti-thrombotic agent. These combined effects make AnxA1Ac2-26 a promising therapeutic candidate for promoting resolution in the context of thrombo-inflammatory diseases/conditions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/11/2473/s1, Figure S1: Effect of FPR antagonists. Mice (C57BL/6) were subjected to vehicle LPS (10 μg/mouse) for 2 h and treated with vehicle (saline), pan FPR antagonist Boc2 (10 μg/mouse) or FPR2/ALX antagonist WRW4 (55 μg/mouse) 20 min prior to light/dye-induced thrombus formation, with time of onset and blood flow cessation times recorded for cerebral (A) arterioles and (B) venules. Data are means <sup>±</sup> SEM of 5–6 mice/group. \$\$\$\$ *<sup>p</sup>* <sup>&</sup>lt; 0.0001 vs. same group for onset time, Figure S2: Onset and blood flow cessation times in mice with/without endotoxaemia. C57BL/6 mice or sickle cell transgenic mice (STM) were subjected to vehicle (saline) or LPS (0.4 mg/kg) for 2 h. A cranial window was performed, and FITC-dextran injected (10 mg/kg of 5%).Mice were then subjected to intravital microscopy and light/dye-induced thrombus formation, with time of onset and blood flow cessation times recorded for cerebral (A) arterioles and (B) venules. Data are means ± SEM of 5–6 mice/group. \* *p* < 0.05, \*\* *p* < 0.01 vs. C57BL/6 saline group. ## *p* < 0.01 vs. STM saline group. \$ *p* < 0.05, \$\$ *p* < 0.0001 vs. same group for onset time, Figure S3: AnxA1Ac2-26 did not affect neutrophil counts in LPS-treated mice. Peripheral blood neutrophil counts were assessed following saline (vehicle) or AnxA1Ac2-26 (4 mg/kg) administration for 20 min following 2 h saline (vehicle) or LPS (0.4 mg/kg) administration. Data are means ± SEM of 5–6 mice/group. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. LPS vehicle (saline), Figure S4: Effect of FPR antagonists in sickle cell transgenic mice (STM). STM were subjected to vehicle LPS (10 μg/mouse) for 2 h and treated with vehicle (saline), pan FPR antagonist Boc2 (10 μg/mouse) or FPR2/ALX antagonist WRW4 (55 μg/mouse) 20 min prior to light/dye-induced thrombus formation, with time of onset and blood flow cessation times recorded for cerebral (A) arterioles and (B) venules. Data are means ± SEM of 5–6 mice/group. \$\$\$\$ *p* < 0.0001 vs. same group for onset time.

**Author Contributions:** S.A.V., E.Y.S., and F.N.E.G. were involved in the conceptualisation of the study and collected and analysed the data. All authors interpreted the data. S.A.V., J.A. and F.N.E.G. wrote the manuscript. F.N.E.G. was the project administrator and acquired funding. All authors critically reviewed the paper and approved its final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** The American Heart Association, grant number 16IRG27790071 and the Royal Society Wolfson Foundation, grant number RSWF\R3\183001 (FNEG).

**Acknowledgments:** The authors would like to thank Professor Robert Hebbel (University of Minnesota) for the STM.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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### *Article* **RNA-Sequencing-Based Transcriptomic Analysis Reveals a Role for Annexin-A1 in Classical and Influenza A Virus-Induced Autophagy**

**Jianzhou Cui 1,2,**†**, Dhakshayini Morgan 1,2,**†**, Dao Han Cheng 1,2, Sok Lin Foo 1,2,3, Gracemary L. R. Yap 1,2,3, Patrick B. Ampomah 1,2, Suruchi Arora 1,2, Karishma Sachaphibulkij 1,2, Balamurugan Periaswamy 4, Anna-Marie Fairhurst 5, Paola Florez De Sessions <sup>4</sup> and Lina H. K. Lim 1,2,3,\***


Received: 30 April 2020; Accepted: 1 June 2020; Published: 4 June 2020

**Abstract:** Influenza viruses have been shown to use autophagy for their survival. However, the proteins and mechanisms involved in the autophagic process triggered by the influenza virus are unclear. Annexin-A1 (ANXA1) is an immunomodulatory protein involved in the regulation of the immune response and Influenza A virus (IAV) replication. In this study, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated protein 9) deletion of ANXA1, combined with the next-generation sequencing, we systematically analyzed the critical role of ANXA1 in IAV infection as well as the detailed processes governing IAV infection, such as macroautophagy. A number of differentially expressed genes were uniquely expressed in influenza A virus-infected A549 parental cells and A549 ΔANXA1 cells, which were enriched in the immune system and infection-related pathways. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the role of ANXA1 in autophagy. To validate this, the effect of mechanistic target of rapamycin (mTOR) inhibitors, starvation and influenza infection on autophagy was determined, and our results demonstrate that ANXA1 enhances autophagy induced by conventional autophagy inducers and influenza virus. These results will help us to understand the underlying mechanisms of IAV infection and provide a potential therapeutic target for restricting influenza viral replication and infection.

**Keywords:** influenza; RNA-sequencing; transcriptomics; autophagy; Annexin-A1

### **1. Introduction**

Influenza A virus (IAV) is a respiratory pathogen which causes widespread infections globally. It is made up of an enveloped capsid that encloses single-stranded RNA. The RNA genome encodes for 13 proteins [1]. Specific viral proteins such as hemagglutinin (HA) and neuraminidase (NA) [2] are found on the surface as antigenic glycoproteins, while others such as matrix1 (M1) and matrix2 (M2) are found on the inside of bilayer lipid membranes [3]. Another viral protein, non-structural protein 1 (NS1), inhibits type 1 interferon (IFN) synthesis and Double-strand RNA (dsRNA)dependent protein kinase R, and thus impedes the host innate response [4,5]. When the virus enters the host cell, the viral life cycle begins. It uses host cell machinery for its replication and transcription [6].

Macroautophagy, microautophagy and chaperone-mediated autophagy are the three different types of pathways involved in autophagy, and all of them require lysosomal degradation [7]. Autophagy plays a role in host defense during pathogenic invasions by preventing viral replication through the removal of pathogenic protein aggregates in the cytoplasm [8]. However, emerging evidence has demonstrated that the autophagy process is regulated by the influenza virus for its benefit [9]. During virus infection, the virus requires the host cells to survive and proliferate and thus it will activate pro-survival mechanisms that include autophagy. Similarly, IAV diverts cell death induced by apoptosis to that of autophagy and this results in prolonged survival and increases virus titers because of enhanced viral replication and deregulation of immune responses [10]. The viral proteins, HA, M2 and NS1, have been reported to be involved in the induction of autophagy by IAV [11]. IAV can inhibit mechanistic target of rapamycin (mTOR) via regulating the mTOR inhibitor tumor suppressor protein 2 (TSC2) [12] and prevents autophagosome fusion with lysosomes via M2 [3], which contains a Microtubule-associated protein light chain 3 (LC3)-interacting domain, causing LC3 to localize to the plasma membrane [13]. This subverted autophagy as fusion was disallowed. Hence, influenza virus triggers the initiation of autophagy but prevents the final steps of autophagosome fusion with lysosomes, utilizing autophagy to accumulate viral components [14].

Briefly, macroautophagy begins with the isolation of the membrane to form a phagophore that is mediated by the unc-51 like autophagy activating kinase 1 (ULK1) kinase complex. Next, Beclin-1 and VSP34, also known as Class III phosphoinositide 3-kinase (PI3K) complex, drives nucleation of the isolated membrane. ATG9 and VMP1 subsequently recruit lipids to the isolated membrane. For the closure of isolated membrane and formation of an autophagosome, the two ubiquitin-like conjugate systems, ATG12-ATG5 and Microtubule-associated protein 1A/1B-light chain 3 (LC3), are involved. In the ATG12-ATG5 system, ATG7 (E1-like enzyme) and ATG10 (E2-like enzyme) are needed to help in the conjugation of ATG12 to ATG5, which is then linked to ATG16. Together the ATG12-ATG5-ATG16 complex forms an E3-like ligase of LC3 and stabilizes the autophagosome [15]. In the LC3 conjugation system, LC3 is cleaved by ATG4 to become LC3-I and LC3-I is converted to LC3-II after being conjugated to phosphatidylethanolamine. Hence, the ATG8/LC3 system plays an essential role in the proper development of autophagic isolation membranes and the biochemical changes of ATG8/LC3 (lipidation and membrane translocation) have been well established as an essential autophagy marker [16].

Annexin 1 (ANXA1), the 37 kDa protein containing 346 amino acids, was the first identified member of the annexin superfamily. It has a structure which consists of a core domain made of alpha helices. The regulatory region is localized at the *N* terminus, which contains sites for phosphorylation and proteolysis [17]. In the presence of calcium, ANXA1 binding to negatively charged phospholipids was mediated by Ca2+-binding motif which located in core domain [18]. ANXA1 was discovered to mediate the anti-inflammatory effect of glucocorticoids, where it inhibits the action of phospholipase A2 (PLA2) by both direct enzyme inhibition and suppression of cytokine-induced activation of the enzyme, limiting the supply of arachidonic acids needed for the synthesis of prostaglandins, thus suppressing inflammation [19].

Although initially discovered in the late 1970s due to its role in inflammation, ANXA1 has also been found to play a role in tumorigenesis, with multiple functions in proliferation, differentiation, apoptosis, migration and invasion [20]. We have recently reported that ANXA1 enhances endosomal trafficking of influenza virus and enhances apoptosis [21]. Furthermore, expression levels of ANXA1 were increased in porcine monocytes during infection with swine flu virus [22], and in human nasal swabs of influenza A virus-infected patients [21].

In this study, we aimed to understand the role of ANXA1 in influenza virus infection more deeply using RNA-sequencing based transcriptomic analysis. Using differential gene expression and gene ontology and subsequent verification experiments, we identified that ANXA1 plays an important role in autophagy induced by classical and viral means.

### **2. Materials and Methods**

### *2.1. Mice*

BALB/c ANXA1−/<sup>−</sup> mice were a kind gift from Prof. Roderick Flower from the William Harvey Research Institute, UK. Mice were age-matched and BALB/c mice were used as control mice for each experiment. All mice were maintained under pathogen-free conditions in the animal housing unit and were transferred to the ABSL2 facility for experiments involving infection with IAV. All animal work was approved by the Institutional Animal Care and Use Committee (Protocol number R13-5101) and followed National Advisory Committee for Laboratory Animals Research (NACLAR) Guidelines on the Care and Use of Animals for Scientific Purposes.

### *2.2. Viruses*

For viral propagation, 1 hemagglutinating unit (HAU) virus A/Puerto Rico/8/1934(H1N1) Influenza A virus (A/PR8) was injected into 10–12-day incubated chicken eggs and further incubated for 3 days. On day 3, the eggs were chilled at −80 ◦C to euthanize the embryo and the allantoic fluid was collected. The fluid was spun in 100,000 molecular weight cutoff (MWCO) concentrators to concentrate the virus and viral plaque assays were performed to quantify the viral titers before use.

### *2.3. Cell Culture*

The human epithelial lung cancer cell line, A549 parental cell line (CCL-185, ATCC, Gaithersburg, MD, USA), A549 ΔANXA1 cells, ATG5 wild-type Mouse Embryonic Fibroblasts (MEFs) and ATG5−/<sup>−</sup> MEFs were cultured at 37 ◦C in a humidified atmosphere with 5% CO2 incubator. The media used for full nutrient and starvation medium were Dulbecco's Modified Eagle Medium (DMEM) and Earle's Balanced Salt Solution (EBSS) respectively.

### *2.4. A549-*Δ*ANXA1 Cell Line Generation*

To produce the A549-ΔANXA1 cell line, clustered regularly interspaced short palindromic repeat-caspase 9 (CRISPR-Cas9) transfection was performed. The plasmid contains the two ANXA1 single guide RNA (sgRNA) (pls2#-CAAACTGTGAAGTCATCCAA and pls4#-ATGCAAGGCAGCGACATCCG were generated by Horizon Discovery Group). The single cell line of A549-ΔANXA1 was isolated using the protocol from the Horizon Company online manual. Generally, A549 cells were seeded in 10 cm dishes and transfected with 10 μg of plasmid per dish using Turbofect, according to the manufacturer's instructions. After 24 h, cells were sorted for positive green fluorescent protein (GFP) expression using the Beckman-Coulter Mo-Flo Legacy Cell Sorter into 96-well plates with 5 cells seeded per well. Cells were expanded to 6-well plates and a preliminary Western blot was conducted to screen for cells with less or no ANXA1 present compared to A549 control cells. Those with less or no ANXA1 present were then seeded again into 96-well plates as single clones and expanded. A secondary Western blot was conducted to determine single cell clones with no ANXA1 present.

### *2.5. Total RNA Extraction, Library Construction and RNA-Sequencing and Data Analysis*

Total RNA was isolated from cells, post treatment, using the RNeasy mini column purification kit (Qiagen, Limburg, The Netherlands) according to the manufacturer's instructions. Cells were washed in 1X PBS on ice, prior to RNA extraction. Total RNA extracts were run on the Agilent bioanalyzer using the eukaryote total RNA pico chips to determine the RNA integrity values (7.4–9.4, with an average of 8.8). TruSeq Stranded mRNA sample preparation was used as per the manufacturer's instructions for next-generation library preparation. Briefly, library preparation entailed: Purification of mRNA using poly-T oligo-attached magnetic beads, fragmentation of mRNA, first and second strand cDNA synthesis, A-tailing and ligation of adapters with multiplex indexes, according to the manufacturer's instructions. Samples were enriched with 15 PCR cycles followed by Agencourt AMPure XP magnetic bead (Beckman Coulter, Brea, CA, USA) clean up according to manufacturer's instructions. Quality of cDNA libraries was checked with Agilent D1000 Tapestation Assay (Agilent 4200 Tapestation System). Next-generation sequencing was performed using Illumina Hiseq 4000 flow cell, 2 × 151 base pair-end runs. PhiX was used as control.

The RNA-Seq transcriptome datasets were mapped to the Genome Reference Consortium Human Build 38 release 86 (GRCh38.r86) by using the Spliced Transcripts Alignment to a Reference (STAR) aligner [23]. Reads that were unambiguously mapped to a given gene were counted using the high-throughput sequencing (HTSEQ)-COUNT tool, available under the HTSeq python framework [24]. These gene-based raw read counts were used for differential gene expression analyses using Bioconductor package EdgeR [25]. Initially, sample read counts were adjusted for library size and normalized using the trimmed mean of m-values (TMM) method. Differential gene expression between groups were assessed using the exactTest method in EdgeR. Genes were called differentially expressed at false discovery rate (FDR) < 0.05. Differentially expressed genes with at least a log2 fold-change of ±1 were considered for further functional annotation.

RNA-Sequencing data was submitted to Sequence Read Archive (SRA). Accession number has been provided (BioProject ID: PRJNA637807).

### *2.6. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) Pathway Analysis*

Gene ontology (GO) enrichment analysis were performed by the Database for Annotation, Visualization and Integration Discovery (DAVID) Functional Annotation (https://david.ncifcrf.gov/home. jsp) and biological pathway analyses were carried out using REACTOME (http://www.reactome.org/ PathwayBrowser). The differentially expressed genes (DEGs) list from two treatment groups: (1) A549 parental cells with or without infection (Group A: wild type (WT) infected versus WT Control) and (2) A549 ΔANXA1 cells with or without infection (Group B: knockout (KO) infected versus KO Control) were selected to perform the GSEA analysis by using WebGestalt (http://www.webgestalt.org/option.php).

### *2.7. Western Blot*

Cells were harvested and centrifuged, and supernatant was collected for protein concentration using a BioRad spectrophotometer or stored at −80 ◦C. Samples were loaded onto 15% (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (SDS-PAGE) gel which was run in 1× running buffer at 120 V for 2 h following the transfer procedure. After which the nitrocellulose membranes were blocked using 3% skimmed milk before primary antibodies were added, and the blots were incubated overnight at

4 ◦C shaking. The secondary antibodies were added and were washed prior to chemiluminescent detection. The primary antibodies used were ANXA1 (sc-12740, Santa Cruz, Dallas, TX, USA), LC3 (# M152-3, MBL, Woburn, MA, USA), Atg3 (#3415, Cell signaling, Danvers, MA USA), β-Actin (#4970, Cell signaling, Danvers, MA, USA) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#5174, Cell signaling, Danvers, MA, USA). The secondary antibodies used were goat anti-mouse Horseradish Peroxidase (HRP) (#sc-2005, Santa Cruz, Dallas, TX, USA) and goat anti-rabbit HRP (#sc-2030, Santa Cruz, Dallas, TX, USA).

### *2.8. Confocal Microscopy*

Coverslips were placed into each well of a 12-well plate. Cells were plated on top of the cover slips and left to incubate with full media and 10 uL of anti-LC3 B GFP antibody (Molecular probes by Life Technologies, Waltham, MA, USA) for 1 h at room temperature and treated with EBSS over various time points. 4% paraformaldehyde (PFA) was added to each well at 4 ◦C and DAPI (4 ,6-diamidino-2-phenylindole) was counterstained for 10 min. The cover slips were washed with PBS 3x and mounted onto microscope slides. The dry slides were visualized with a confocal microscope (Leica LSM 510, Wetzlar, Germany).

### *2.9. Influenza A Virus (IAV) Infection*

Forin vitro experiments, cells were plated in full media overnight, and washed with PBS. The inoculum containing 1 MOI (Multiplicity of infection) of HIN1 virus in serum-free medium was incubated with the cells according to the various treatment timings. After which, the media was aspirated, and the cells were washed with PBS. For in vivo experiments, WT and ANXA1−/<sup>−</sup> Balb/c mice were infected with 500 plaque-forming unit (pfu) of influenza virus intratracheally. On days 0, 1, 3 and 5 post-infection, the mice were sacrificed, and their lung lysates were obtained for Western blot analysis.

### *2.10. Statistical Analysis*

Data shown are the mean ±standard error of the mean (SEM) of 3–5 independent experiments for in vitro work and 3–5 mice for mice work. Student's unpaired T test and two-way analysis of variance (ANOVA) for grouped data with Bonferonni's multiple comparison tests were performed for all datasets. GraphPad Prism (GraphPad software, San Diego, CA, USA) was used for analysis.

### **3. Results**

### *3.1. Detection of CRISPR-Cas9-Induced ANXA1 Mutations in A549* Δ*ANXA1 Cells*

First, to study the role of ANXA1 in the context of IAV infection, ANXA1 was deleted from A549 cells using Crispr-Cas9 technology. Two gRNA sequences that target ANXA1 were custom designed by and obtained from Horizon Discovery. The plasmid-encoding gRNAs that target ANXA1 also encode for GFP, which is expressed once the plasmid has been transfected into the cell. The plasmids were transfected into A549 cells separately and cells positive for GFP were sorted using the Beckman-Coulter Mo-Flo Legacy Cell Sorter into 96-well plates. Single cell clones were expanded, and validation experiments were performed to confirm the deletion. Mutation of ANXA1 in sgRNA #4 regions located in domain I introduced a frameshift mutation which translated into a truncated ANXA1 protein. The off-target effects of #4 sgRNA were also analyzed by online tool, COSMID (https://crispr.bme.gatech.edu/) [26] (Supplemental Table S1). Our sequencing results revealed that no off-target mutations were detected at any of the off-target sites.

Transcript Expression Profiling in Influenza Virus-Infected A549 Parental and ΔANXA1 Cells

To understand the global and general transcripts expression profiles after IAV replication, and the importance of ANXA1 in this process, we performed global RNA-Sequencing (RNA-Seq) in A549 parental

and A549 ΔANXA1 cells with and without IAV infection: (1) A549 parental cells with or without infection (Group A: WT infected versus WT Control) and (2) A549 ΔANXA1 cells with or without infection (Group B: KO infected versus KO Control). After normalizing read count data and setting the significance and fold-change (*p* < 0.01 and log2 fold-change ≥ |1|), transcripts were significantly changed or differentially expressed (DE) and are listed in the Supplemental Table S2.

When A549-infected versus A549 control samples were compared, a total of 1498 differentially expressed transcripts were detected, of which included 1239 transcripts (82.7%) that were significantly upregulated and 259 transcripts (17.3%) which were significantly downregulated (*p* < 0.05) (Figure 1A). A549-ΔANXA1-infected cells were compared against A549-ΔANXA1-uninfected controls, and a total of 1302 differentially expressed transcripts were detected, and globally significant transcript expression patterns were plotted on a volcano map, illustrating that 90.9% of transcripts were upregulated while the remaining 9.1% were downregulated (Figure 1B). Three gene transcripts, MDM2 Binding Protein (MTBP), Pro-Melanin Concentrating Hormone (PMCH) and Fat mass and obesity-associated (FTO) were downregulated both in A549 parental and A549-ΔANXA1 cells, together with 626 other transcripts which were upregulated in both cell types, indicating that these transcripts are ANXA1-independent (Figure 1C). Two sets of transcripts which are uniquely expressed in A549-infected and A549-ΔANXA1-infected cells are listed in Supplemental Table S2 and globally expressed on volcano plots in Figure 1D,E. 605 upregulated transcripts and 256 downregulated transcripts were uniquely expressed in parental A549 cells infected with IAV, while 557 upregulated transcripts and 108 downregulated transcripts were uniquely expressed in A549-ΔANXA1 cells infected with IAV.

### *3.2. Gene Ontology (GO) and GSEA Pathway Analysis*

The transcripts which were commonly expressed in both WT (A549 parental cells) and KO (A549-ΔANXA1) infected cells, as well as the transcripts which were uniquely expressed in both cell types, were analyzed further with gene ontology, focusing on biological process pathways. The top enriched fractions are shown in Figure 2 (*p* < 0.05) and Supplemental Table S3. For commonly expressed transcripts (red bars), representing ANXA1-independent genes, infection of IAV resulted in a high enrichment of the biological processes relating to the response to Redox state (GO0051775), regulation of cellular senescence (GO2000772) and negative regulation of wound healing (GO0061045). In unique transcripts, expressed uniquely in the A549 parental infected cells and not the A549-ΔANXA1 cells (blue bars), representing genes which require ANXA1 either to be expressed or repressed, the most enriched terms are the response to type-1 interferons (GO0035455/6), and the regulation of viral genome replication (GO045070/1), which we have shown previously to be regulated by ANXA1 [21]. Of interest, macroautophagy (GO0016236) is also a biological process which is highly enriched and requires ANXA1.

For unique transcripts which are expressed in A549-ΔANXA1-infected cells and not A549 parental cells (green bars), representing genes which can be regulated by ANXA1, the enriched biological processes include positive regulation of apoptosis (GO1900119), regulation of mitochondrial apoptosis (GO1900740) and actin cytoskeleton organization (GO0030036), which have also been shown previously to be regulated by ANXA1 [21]. The global distribution pattern for the top enriched pathway in each group is presented in Supplementary Figure S1.

Using DAVID (https://david.ncifcrf.gov/home.jsp), we looked closer into the genes which are involved in immunity and viral responses in both cell types (Figure 3A), and Interferon Induced Protein With Tetratricopeptide Repeats 1(IFIT1), IFIT2 and IFIT3, (also known as interferon stimulated gene factor 56 (ISG56), ISG54 and ISG60, respectively), which are all interferon-induced anti-viral proteins, are increased in A549 WT but not A549-ΔANXA1 cells, signifying that ANXA1 is required for these genes. In addition, Tripartite motif-containing protein 5 (TRIM5) and TRIM56, which are tripartite motif-containing Ring-type

E3-ubiquitin protein ligases, which are also anti-viral proteins, also require ANXA1 to be expressed. Autophagy-related proteins' expression, which are controlled by ANXA1, include ATG5, ATG2B and other genes involved in protein trafficking in endosomes and lysosomes, such as vacuolar protein sorting 51 (VPS51) and VPS16 (Figure 3B). This is interesting as we have previously shown that ANXA1 can enhance endosomal trafficking of the virus [21]. Transcription factor XBP1 and serine threonine kinase STK11, which are also dependent on ANXA1 for expression after influenza infection, have both been shown to be important in the regulation of autophagy [27–30]. Therefore, we validated this finding in vitro in lung epithelial cells.

**Figure 1.** Global overview of the RNA-Sequencing data of influenza A virus (IAV)-infected lung epithelial (A549) cells with or without ANXA1 deficiency. (**A**,**B**) Volcano map showing the significant differentially expressed transcripts in A549 and A549-ΔANXA1. (**C**) The distribution abundance of differentially expressed genes/transcripts in upregulated and downregulated pattern in group A and B. MTBP (MDM2 Binding Protein), PMCH (Pro-Melanin Concentrating Hormone), FTO (alpha-ketoglutarate-dependent dioxygenase). (**D**,**E**) The unique expressed transcripts in group A and group B are plotted on a volcano map. The top genes selected showing the highest fold-changes are presented as red dots.

To further identify the potential function and pathway enrichment of DEGs in the two groups, GSEA was conducted to search significant pathways enriched in the highly expressed DEGs in both upregulation and downregulation genes (Supplementary Table S2). The top ten enriched gene sets in both up- and down-regulated expression pattern for Group A (WT infected versus WT Control) are shown in Supplementary Figure S2B. Two of the gene sets, i.e., plasma lipoprotein assembly, remodeling, and clearance, and Transcriptional Regulation by TP53 were enriched in upregulated DEGs (FDR > 0.05), while Viral mRNA Translation and Influenza Viral RNA Transcription and Replication were enriched in downregulated DEGs (FDR < 0.05) in group A (Supplementary Figure S2A). As for group B (KO infected versus KO Control), we found that gene sets of Regulation of Hypoxia-inducible Factor (HIF) by oxygen and Infectious disease were enriched in upregulated DEGs (FDR > 0.05), while Fc epsilon receptor (FCERI) signaling and Transport to the Golgi and subsequent modification were enriched in downregulated DEGs (FDR < 0.05) (Supplementary Figure S2B).

### *3.3. IAV Enhances Autophagy through Regulation of Autophagic Proteins*

We first validated the role of autophagy in influenza virus infection. The transcription of autophagy-related genes Beclin-1 (BECN1, ATG6) and Autophagy-related gene 3 (ATG3) were determined after IAV infection at 4 and 24 h in A549 lung epithelial cells. BECN1 and ATG3 mRNA were significantly increased after 24 h post-infection (hpi) as compared to their respective uninfected controls (Figure 4A,B). Successful viral infection can be observed from the significant increase in non-structural protein 1 (NS1) viral mRNA expression after infection (Figure 4C). Autophagy-related proteins BECN1 and ATG3 were similarly higher 24 h after IAV infection, together with an increase in LC3-II (Figure 4D), indicating an induction of autophagy after IAV infection. To study the role of autophagy deficiency on the viral replication, the effect of ATG5 deficiency on gene expression of NS1 and M2 were examined using ATG5

WT and ATG5−/<sup>−</sup> MEFs. After 24 h of infection, both NS1 and M2 gene expression were significantly lower in ATG5−/<sup>−</sup> MEFs compared to WT MEFs (Figure 4E), suggesting that autophagy is important for viral RNA synthesis, although ATG5-deficient cells may also undergo more apoptosis. Taken together, these results indicate that autophagy is important for influenza virus infection and viral RNA synthesis.

**Figure 3.** The specific genes involved in given gene ontology for uniquely expressed up and down genes in A549 (WT) and A549-ΔANXA1 (KO) infected versus control samples. Specific genes are highly enriched and involved in immunity and viral responses (**A**), autophagy (**B**) and translational regulation (**C**).

### *3.4. Silencing ANXA1 Results in the Reduction of Autophagosomes and Autophagy after Starvation and Inhibition of mTOR*

Starvation or low nutrient levels triggers autophagy via various signaling pathways, including Tuberous sclerosis 1 (TSC1)-mTOR, recombination-activating genes (RAGs) and AMP-activated protein kinase (AMPK) pathways [31]. Our previous studies demonstrate that ANXA1 is important in IAV infection and apoptosis [21]. Thus, to determine if ANXA1 is important in IAV-dependent autophagy, A549 parental and A549-ΔANXA1 cells were first treated with EBSS media to induce starvation and

autophagy for increasing time points. The expression of LC3II was observed to be significantly inhibited in A549-ΔANXA1 cells at the later time points when treated with EBSS (Figure 5A,B).

**Figure 4.** IAV infection increases autophagy-related genes in lung epithelial cells. (**A**–**C**) A549 lung epithelial cells were infected with 1 MOI of PR8 and reverse transcription PCR (RT-PCR) was performed for mRNA analysis of autophagy-related genes BECLN1, ATG3 and virus protein gene non-structural protein 1 (NS1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for all quantitative RT-PCR analyses. Data are representative of 3–6 SEM of lung epithelial cells. \* *p* < 0.05, \*\* *p* < 0.01, against uninfected cells. (**D**) Western blot analysis of autophagy-related proteins BECN1, ATG3 and LC3-II after IAV infection in A549 cells. (**E**) WT MEF and Atg5−/<sup>−</sup> MEF cells were infected with 1 MOI of H1N1 influenza. Total RNA was isolated from infected cells at 12 h post-infection. The mRNA level of viral NS1 and M2 were determined by using real-time CR. Results are representative of three independent experiments.

In addition, confocal staining was performed to detect autophagosomes labelled by anti-LC3 green fluorescent protein (GFP) formed with a long period of starvation. As illustrated in Figure 5C, autophagosome formation was increased in A549 cells but not A549-ΔANXA1 cells after 24 h, with a significant difference in the number of autophagosomes formed per cell (Figure 5D). This suggests that ANXA1 may be important in the regulation of autophagosome formation. MTOR negatively regulates the ULK1 kinase complex and thus inhibits autophagy [28,29]. Therefore, rapamycin, an inhibitor of mTOR [30,31], and Torin-1 (Supplementary Figure S3A,B) were used to induce autophagy. In addition, an autophagy inhibitor, 3-Methyladenine (3-MA), was used together with rapamycin. 3-MA inhibits class III PI3K and thus inhibits autophagy by blocking autophagosome formation (Figure 5E). After 24 h, LC3-II was only expressed in A549 WT cells treated with rapamycin alone and rapamycin with 3-MA,

or Torin-1. However, no LC3-II expression was detected in A549-ΔANXA1 cells treated with the same stimuli, indicating that the mTOR inhibitor-induced autophagic flux may be ANXA1-dependent.

**Figure 5.** mTOR inhibitors and starvation induce autophagy in A549 but not in A549-ΔANXA1. (**A**,**B**) A549 cells and A549-ΔANXA1 cells were treated with EBSS over various time points (0, 2, 4, 6 and 8 h). The cell lysates were then collected and a Western blot analysis using LC3 antibody was performed. (**C**,**D**) The number of autophagosomes (labeled by anti-LC3-GFP) was counted in A549 cells and A549-ΔANXA1 cells after being treated with EBSS at various time points (0, 8, 16 and 24 h). The autophagosomes were labelled using anti-LC3-GFP (green) and the nuclei of the cells were stained using DAPI (blue). Autophagosomes were counted per cell in at least 3–5 cells per view, and the error bars and significance data were generated from 3 fields of view. (**E**) Western blot analysis was performed after cells were treated with 200 nM of rapamycin and 0.75 mg/mL of 3-Methyladenine (3-MA) for 24 h. Data is representative of three independent experiments. \* 0.01 < *p* < 0.05, \*\* *p* < 0.01. Scale bars = 25 μm in all panels.

### *3.5. ANXA1 Is Involved in Autophagy Induced by Influenza Virus In Vitro and In Vivo*

Next, to investigate if ANXA1 plays a role in the intracellular degradation process induced by IAV infection, both A549 cells and A549-ΔANXA1 cells were infected with H1N1 PR8 IAV at multiplicity of infection (MOI) 1 over various time points. Upon treatment with IAV, LC3-II expression increases and is significantly higher in A549 cells but not A549-ΔANXA1 cells after 8 h of infection (Figure 6A,B). In addition, to further investigate the role of ANXA1 in IAV-induced autophagy flux, chloroquine (CQ) was used to treat the A549 and A549-ΔANXA1 lung cancer cells with or without IAV infection. IAV infection increased the autophagy flux in A549 cells but not in A549-ΔANXA1 cells, indicating that deletion of ANXA1 can suppress IAV-induced autophagic flux (Figure 6C,D).

**Figure 6.** ANXA1 is involved in autophagy induced by influenza virus. (**A**,**B**) Cells were infected with H1N1 at 1 MOI over various time points (0, 2, 4, 8 and 10 h). Quantification of LC3-II expression using densitometric analysis. Data represent two independent experiments (*p* < 0.05). (**C**,**D**) A549 and A549-ΔANXA1 cells were infected with H1N1 at 1 MOI over 12 h, CQ 50 uM for 2 h. Quantification of LC3-II expression using densitometric analysis \*\* *p* < 0.01. (**E**, **F**) WT and ANXA1−/<sup>−</sup> Balb/c mice were infected with 500 pfu of influenza virus intratracheally. On days 0, 1, 3 and 5 post-infection, the mice were sacrificed, and their lung lysates were obtained for Western blot analysis. \* 0.01 < *p* < 0.05.

Having shown that ANXA1 plays a positive role in influenza virus-induced autophagy in vitro, we next replicated the results in vivo. WT mice and ANXA1−/<sup>−</sup> mice were infected with influenza virus at 500 plaque-forming units (pfu). At day 1, day 3 and day 5 post-infection, these mice were sacrificed, and lung lysates were obtained and used to determine the expression of LC3-II after IAV infection. As illustrated in Figure 6E,F, the expression of LC3-II appeared prominently only for WT mice and very little was observed for ANXA1−/<sup>−</sup> mice, indicating that ANXA1 is indeed required to induce autophagy triggered by IAV.

In conclusion, our results show that ANXA1 can enhance autophagy induced by conventional autophagy inducers as well as influenza virus.

#### **4. Discussion**

In this study, transcriptomic RNA-Seq was used to investigate the role of ANXA1 in IAV infection and validated experiments show that ANXA1 is important in classical and IAV-induced autophagy. The abundance and significant over-representation of various transcripts were found in cells expressing and silenced for ANXA1 using CRISPR/Cas9. Our results show unique genes which require ANXA1 to be expressed (WT unique up), genes which require ANXA1 to be suppressed during IAV infection (WT unique down), as well as genes which can be regulated positively (KO unique down) or negatively (KO unique up) by ANXA1, but where ANXA1 is not critical for expression. The highest hit for genes requiring ANXA1 to be expressed belong to pathways related to "immunity and virus response", including innate immune response and responses to the virus and anti-viral defense. In contrast, genes requiring ANXA1 to be suppressed during IAV are those shown to be involved in viral transcription and translation, including genes which are highly enriched in translational regulation, such as RPS3, RPL23, RPL24 and RPL25. These are ribosomal proteins involved in influenza virus RNA transcription and viral mRNA translation. Unique genes upregulated after IAV infection in A549-ΔANXA1 cells but not in WT A549 cells, which are genes which can be negatively regulated by ANXA1, are enriched mostly in mitochondria and mitochondrial function. This includes enzymes such as HAGH (Hydroxyacyl glutathione Hydrolase), POLRMT (RNA polymerase in Mitochondria), ACSM4 (Acyl-CoA Synthetase), LIAS (Lipoic Acid Synthetase) and COX7A (Cytochrome c oxidase). This suggests that ANXA1 can regulate many enzymes involved in lipid metabolism and mitochondrial metabolism. This demonstrates a multi-pronged function of ANXA1, where ANXA1 is required for the expression of anti-viral genes in response to type-1 interferons, similar to what has been described previously [32]. In contrast, genes requiring ANXA1 to be suppressed during IAV are those shown to be involved in viral transcription and translation. This demonstrates a multi-pronged function of ANXA1, where ANXA1 is required for the expression of anti-viral genes in response to type-1 interferons, yet is also required for the suppression of genes important in virus transcription and translation. ANXA1 also is required for the expression of genes relating to endosomal trafficking and autophagosome assembly, which can be linked to our previous work, showing that ANXA1 promotes virus replication through enhancement of endosomal trafficking [21]. Genes which ANXA1 can positively regulate are centered around cell adhesion and neuronal development, while genes which ANXA1 can negatively regulate are mostly related to mitochondrial function and cell metabolism. As ANXA1 has been shown to enhance virus replication in vitro and in vivo [21], it may be possible to predict that the roles of ANXA1 in endosomal trafficking and the regulation of mitochondrial function may have more impact on virus replication than the regulation of anti-viral immune responses.

While autophagy is shown to promote IAV replication and apoptosis [33,34] and various viral proteins such as nucleoprotein (NP) and M2 can induce the AKT-mTOR autophagy pathway [35], M2 has also been shown to stimulate the initial phase of autophagosome formation, but inhibits autophagosome–lysosome fusion, resulting in the inhibition of anti-apoptotic macroautophagy, a strategy

to enhance its pathogenicity [3]. Enhanced host cell death could limit virus-specific host responses and cytokine production. Whether the stimulation of autophagy is a host-response to viral infection, or the strategy of the virus against the host itself, is yet to be determined. Nevertheless, autophagy and autophagic cell death is essential in influenza replication and pathogenesis. In addition, annexin–pathogen interactions have been well elaborated recently and show that IAV utilize ANXA1 to regulate the host innate immune responses [21,36]. We recently showed that ANXA1 enhances influenza virus infection and viral replication by enhancing cell death and apoptosis [21]. Using RNA-Seq and in vitro and in vivo validation, our study establishes that ANXA1 can enhance autophagy and is crucial for influenza virus-induced autophagy. However, this is not in line with a recent study which demonstrated that silencing of ANXA1 with siRNA significantly inhibited starvation-induced autophagic degradation as measured by the level of Sequestosome 1 (SQSTM1 or p62), although the specific siRNA did not alter starvation-induced LC3-II level [37]. In our study, the expression of LC3-II was higher in A549 parental cells compared to A549-ΔANXA1 cells when treated with EBSS and other autophagy inducers. This discrepancy may be due to the fully functional silencing of ANXA1 via CRISPR-Cas9, or cell-type specific differences. These results suggest that the presence of ANXA1 is important in autophagy triggered by starvation, mTOR inhibitors and influenza virus.

Overall, the data in this study establishes the positive role of ANXA1 in virus-induced autophagy as well as autophagy triggered by other mechanisms that include starvation or inhibitors of mTOR. Hence, the presence of ANXA1 directly benefits viral replication through the enhancement of autophagy, another mechanism through which ANXA1 can act to enhance virus propagation.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/6/1399/s1: Figure S1: Torin 1 induces autophagy in a time-dependent manner in A549 but not in A549. Figure S2: Pathways involved in the response caused by IAV infection in A549 (WT) and A549 ΔANXA1 (KO) infected cells. Figure S3: Gene Set Enrichment Analysis (GSEA) analysis in IAV infected A549 WT and A549 ΔANXA1 cells. Table S1: Off-target effects of #4 CRISPR plasmid, Table S2: Differentially expressed genes, Table S3: Gene Set Enrichment Analysis.

**Author Contributions:** Conceptualization, D.M., S.A. and L.H.K.L; Data curation, J.C.; Formal analysis, D.M., P.B.A., D.H.C., G.L.R.Y. and J.C.; Funding acquisition, L.H.K.L.; Investigation, J.C., D.M., S.L.F., G.L.R.Y., K.S., B.P. and P.F.D.S.; Methodology, J.C., S.L.F., S.A. and B.P.; Resources, B.P., A.-M.F. and P.F.D.S.; Supervision, L.H.K.L.; Writing—original draft, D.M.; Writing—review and editing, J.C. and L.H.K.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants from the National Medical Research Council in Singapore (NMRC/CBRG/056/2014) awarded to L.H.K.L., F.S.L. and G.Y. were supported by a scholarship from the NUS Graduate School of Science and Technology.

**Acknowledgments:** We would like to thank Roderick Flower for providing the ANXA1−/<sup>−</sup> mice.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**




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