PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke

Neutrophils are key effector cells in the pathogenesis of stroke. Using in vivo murine models, Dhanesha and colleagues identify the multifunctional protein pyruvate kinase muscle 2 (PKM2) in neutrophils as a key modulator of outcome. Induction of stroke results in nuclear translocation of PKM2 in neutrophils driving a thromboinflammatory reaction through STAT3 signaling that exacerbates the severity of cerebral ischemia-reperfusion injuries, a process that is potentially amenable to timely, targeted pharmacological inhibition.


Abstract
There is a critical need for cerebroprotective interventions to improve the suboptimal outcomes of patients with ischemic stroke treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke both in humans and in mice. Therefore, we determined the role of PKM2 in stroke pathogenesis utilizing murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2 -/mice on wild-type (PKM2 fl/fl LysMCre + ) and hyperlipidemic background (PKM2 fl/fl LysMCre + Apoe -/-). Controls were littermate PKM2 fl/fl LysMCreor PKM2 fl/fl LysMCre -Apoe -/mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps following cerebral ischemia/reperfusion, suggesting PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot/rtPA stroke models, irrespective of sex, deletion of PKM2 in myeloid cells either in wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced postischemic cerebral thrombo-inflammation (intracerebral fibrin(ogen), platelet (CD41-positive) deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes following stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery following reperfusion.

Introduction
At present, an acute ischemic stroke is managed by intravenous thrombolysis with recombinant tissue plasminogen activator (rtPA) and/or mechanical thrombectomy. While both of these approaches are effective, they have limitations. For example, early arterial re-occlusion and the more unsatisfactory long-term outcome were observed in nearly 17-34% of stroke patients after rtPA administration, 1,2 suggesting modest efficacy of intravenous thrombolysis. While mechanical thrombectomy is much more efficacious; ~ 50% of treated acute stroke patients with large vessel occlusion have suboptimal outcomes. 3 Altogether, these limitations highlight the critical need for novel ancillary treatment that effectively enhances the limited success of stroke reperfusion therapies.
Since ischemic brain injury is aggravated by both thrombosis and inflammation (thromboinflammation), 4,5 an ideal target to improve stroke outcome should be one that inhibits thromboinflammatory responses without a significant risk of bleeding complications. Recently, the glycolytic enzyme pyruvate kinase muscle 2 (PKM2) has been implicated not only as a critical regulator of aerobic glycolysis but also as an activator of transcription of pro-inflammatory mediators, including IL-1 and IL-6. [6][7][8] Pyruvate kinase (PK) exists in 4 different isoforms (PKR, PKL, PKM1, PKM2,) and is encoded by 2 distinct genes, PKLR and PKM, in mammals. PKR is expressed in erythrocytes; PKL in liver and kidney; PKM1 is expressed in differentiated adult tissues with high ATP requirement such as heart, brain, and muscle. PKM2 is expressed in many tissues including spleen, lung and all cancer cell lines. 9 During past years, PKM2 has generated significant interest because of its upregulation in activated immune cells, smooth muscle cells, and platelets. [10][11][12][13] Unlike other isoforms of PK, that exist and function as tetramers, PKM2 exists in tetrameric and dimeric forms composed of identical monomers but with different biological activities. In addition to its role in glycolysis, PKM2 also possesses protein kinase activity. 14,15 Upon stimulation, dimeric PKM2 translocate to the nucleus, where PKM2 catalyzes the transfer of phosphate from PEP to serine, threonine, or tyrosine residues on target substrates. [15][16][17][18] The dimeric PKM2 is known to promote inflammatory macrophage activation, 12,19 autoimmune encephalomyelitis, 20 as well allergic airways disease. 7 Neutrophils, the most abundant white blood cells, are among the first cells in the blood to respond to an acute ischemic insult and play a key role in stroke exacerbation. [21][22][23][24][25][26][27] However, whether PKM2 promotes neutrophils hyperactivation upon acute ischemic stroke and thereby mediates ischemic brain injury remains unclear. In the current study, we elucidated the role of PKM2 in the pathogenesis of ischemic stroke. To enhance the translational significance of this study, we specifically measured the effect of sex, preexisting comorbidities, and the two forms of reperfusion (filament mechanical occlusion and autologous clot/rtPA).

MATERIALS AND METHODS
Detailed information on materials and methods is available in the online-only data supplement. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Human subjects
The study involving human subjects was previously approved by the institutional review board at the University of Iowa, and informed consent was obtained from patients or their surrogates.

Filament and embolic stroke models
Mice were anesthetized with 1-1.5% isoflurane mixed with medical air. After a midline incision, the right common carotid artery was temporarily clamped, and a silicon monofilament (Doccol catalog# 702245PK5re) or a single homologous embolus (~15 mm) was inserted via the external carotid artery into the internal carotid artery up to the origin of the middle cerebral artery.
Reperfusion was achieved by removing the filament after 60 or 30 minutes and opening the common carotid artery (in case of filament model) or by infusion of rtPA (10 mg/kg, 10% volume by bolus and remaining slow infusion for 30 minutes) in case of embolic model. Laser Doppler flowmetry was used for each mouse to confirm the successful induction of ischemia and reperfusion.

Results
Human and mouse circulating neutrophils exhibit increased PKM2 nuclear translocation following acute ischemic stroke Evidence suggests increased PKM2 nuclear translocation in cancer cells and in the activated immune cells when stimulated with agonists. 6,12,16,19 We determined whether PKM2 nuclear translocation increases in neutrophils in the setting of ischemic stroke. Western blot analysis revealed a  3-fold increase in nuclear PKM2 levels in peripheral neutrophils of patients with ischemic stroke treated with mechanical thrombectomy compared with healthy controls ( Figure   1A). The baseline characteristics of the patients and recanalization status are shown in Table S1.
Similarly, a time-dependent increase (up to 6 hours) in nuclear PKM2 expression was observed in peripheral neutrophils isolated from the wild-type (WT) mice that underwent 60 minutes of cerebral ischemia followed by 3, 6, or 23 hours of reperfusion ( Figure 1B). On the other hand, peripheral monocytes did not exhibit increased nuclear PKM2 levels following stroke in mice ( Figure S2).

PKM2 promotes neutrophil hyperactivation following ischemic stroke in mice
We evaluated inflammatory status in the peripheral monocytes and neutrophils of WT mice at an early time point (6 hours) after reperfusion. ELISA revealed increased levels of inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in both monocytes and neutrophils (P<0.05 versus sham, Figure S3). Notably, a marked increase in TNF-α, IL-1β, and IL-6 levels was observed in neutrophils when compared to monocytes following stroke ( Figure S3). We focused on neutrophils because nuclear PKM2 was upregulated in peripheral neutrophils but not in monocytes at 6 hours post-stroke (Figure 1 and Figure S2). Increased pro-inflammatory cytokines status was associated with increased neutrophil extracellular traps (NETs) ( Figure S4A) and expression of several proinflammatory genes, including MPO, elastase, HIF1, and P65 ( Figure S4B), suggesting that cerebral ischemia/reperfusion promotes neutrophil hyperactivation. To confirm a definitive role of PKM2 in neutrophil hyperactivation in the context of ischemic stroke, we generated novel myeloidspecific PKM2-deficient mice (PKM2 fl/fl LysMCre +/-, Figure S1A). Genomic PCR confirmed the presence of LysMCre gene in PKM2 fl/fl mice ( Figure S1B). By Western blotting, we confirmed the absence of PKM2 in neutrophils from PKM2 fl/fl LysMCre +/mice ( Figure 1C). To simplify, from here, littermate PKM2 fl/fl LysMCre -/will be referred as PKM2 fl/fl mice. Next, we subjected PKM2 fl/fl LysMCre +/and littermate control PKM2 fl/fl mice to 1-hour of cerebral ischemia and 6hours of reperfusion. Because upregulated pro-inflammatory cytokines status was associated with increased NETosis in the setting of ischemic stroke, we determined whether PKM2 modulates NETosis by aggravating pro-inflammatory response. Peripheral neutrophils isolated 6 hours postreperfusion were stimulated with a suboptimal concentration of PMA (10 ng/mL). The percentage of NETs positive-cells was reduced in PKM2 fl/fl LysMCre +/mice (P<0.05 versus PKM2 fl/fl mice, Figure 1D). Furthermore, we found significantly reduced level of inflammatory cytokines TNF-α, IL-1β, and IL-6 and reduction in expression of pro-inflammatory genes, including MPO, elastase, HIF1, IL-1, and p65 in peripheral neutrophils following 6 hours of reperfusion in PKM2 fl/fl LysMCre +/mice (P<0.05 versus PKM2 fl/fl mice, Figure 1EF). Together, these results suggest that PKM2 potentiates neutrophil hyperactivation following ischemic stroke.

Myeloid cell-specific PKM2 -/mice exhibit reduced infarct area and improved long-term sensorimotor outcome
To evaluate the role of PKM2 in stroke outcomes, PKM2 fl/fl LysMCre +/and littermate PKM2 fl/fl male and female mice were subjected to 1-hour of ischemia and 23-hours of reperfusion in the filament model. Both male and female PKM2 fl/fl LysMCre +/mice exhibited smaller infarcts and better neurological outcomes on day 1 when compared with littermate controls (Figure S5). Infarcts and neurological scores were comparable between PKM2 +/+ LysMCre + and PKM2 fl/fl mice ( Figure   S6), ruling out nonspecific effects of LysM-Cre recombinase expression on stroke outcome. Current Stroke Therapy Academic Industry Roundtable (STAIR) guidelines for preclinical assessment of novel therapeutic targets for stroke recommend evaluation of underlying mechanisms for stroke progression, with an assessment of response to treatment in at least two different stroke models in both sexes with preexisting comorbidities that adequately mimic the human physiology. 29,30 Following STAIR recommendations, we generated PKM2 fl/fl LysMCre +/mice and littermate control PKM2 fl/fl LysMCre -/mice on the hyperlipidemic apolipoprotein E-deficient (Apoe -/-) background. We chose the preexisting comorbid condition hyperlipidemia because it is known to exacerbate ischemic damage and worsen the sensorimotor deficit by promoting endothelial dysfunction, inflammation, oxidative stress, and neuronal death, 31 and thereby enhancing stroke sensitivity. All the mice were fed a regular chow diet after weaning until 8 to 10 weeks, an age at which no significant vascular lesions are found (not shown) to minimize the potential confounding effects of advanced atherosclerotic lesions that can impair collateral flow and indirectly influence the stroke outcome. Bodyweight, plasma cholesterol, triglycerides, and complete blood counts were comparable between these groups (Table S2 and S3).
Using the filament stroke model, susceptibility to cerebral ischemia/reperfusion injury was evaluated following 1, 3, and 7 days of reperfusion (Figure 2A). We observed significantly reduced infarct area in PKM2 fl/fl LysMCre +/-Apoe -/mice at day 1 (P<0.05 versus PKM2 fl/fl Apoe -/mice; Figure 2B). Consistent with these results, at day 7, PKM2 fl/fl LysMCre +/-Apoe -/mice exhibited a better survival rate (≈70%) compared to PKM2 fl/fl Apoe -/mice ( Figure 2C). Next, using the same set of mice, we evaluated the modified neurological severity score (mNSS) based on spontaneous activity, symmetry in limb movement, forepaw outstretching, climbing, body proprioception, and responses to vibrissae touch (on the scale of 3-18; higher score indicates a better outcome) and motor function using an accelerated rota-rod test. We observed that PKM2 fl/fl LysMCre +/-Apoe -/mice exhibited improved neurological outcome and motor function on days 1, 3, day 7 compared to PKM2 fl/fl Apoe -/mice (Fig. 2DE). Laser Doppler flow measurements (Table S4) and physiological parameters (Table S5) were similar among groups before, during, and after ischemia. No gross differences in cerebrovascular anatomy were observed between groups Since higher mortality was observed in hyperlipidemic mice after 1 hour of ischemia, we could not determine long-term outcomes up to 4 weeks. Therefore, we reduced ischemia time from 60 minutes to 30 minutes and evaluated sensorimotor recovery up to 4 weeks ( Figure 3A). Consistent with the results of 60 minutes ischemia, PKM2 fl/fl LysMCre +/-Apoe -/mice exhibited reduced infarct area and improved neurological outcome (mNSS) from 1-week up to 4-weeks (P<0.05 versus PKM2 fl/fl Apoe -/-, Figure 3BC). Next, we performed a cylinder test, a sensorimotor test to assess asymmetry in forelimb use during vertical exploratory behavior inside a glass cylinder. We found  Figure 3G). Together, these results suggest that deletion of PKM2 in myeloid cells enhances the post-stroke long-term sensorimotor recovery.
Apoe deficiency in mice promotes blood-brain barrier breakdown and neuronal death, disrupts cerebrovascular reflexes, and worsens ischemic perfusion defect. 31,32 To rule out the role of Apoe deficiency in PKM2-dependent worsening of stroke outcome, we evaluated stroke outcome in another hyperlipidemic model, low-density lipoprotein receptor-deficient (Ldlr -/-) mice using bonemarrow transplantation approach (Figure S8A). Complete blood counts did not differ between the groups. (Table S6). We observed significantly improved stroke outcome in Ldlr -/mice that were transplanted with bone-marrow cells of PKM2 fl/fl LysMCre +/mice when compared with Ldlr -/mice that were transplanted with bone-marrow cells of control PKM2 fl/fl mice ( Figure S8BC).

Myeloid-specific PKM2 -/mice exhibited improved local cerebral blood flow, reduced postischemia/reperfusion thrombo-inflammation
To determine whether improved stroke outcome in the PKM2 fl/fl LysMCre +/-Apoe -/mice was associated with reduced post-ischemia/reperfusion cerebral thrombosis and improved local cerebral blood flow (CBF), laser speckle imaging was performed at different time points. We found that regional CBF was improved at 30, 60, and 120-minutes following reperfusion in  Figure S9AB). Human studies and experimental stroke models suggest that monocytes are recruited into the ischemic area of brain, most abundantly at days 3-7 poststroke. Therefore, we analyzed brain monocyte/macrophage content at day 3 poststroke. Using immunohistochemistry, we found reduced macrophage content (CD68-positive cells) in infarcted regions of PKM2 fl/fl LysMCre +/-Apoe -/when compared with control PKM2 fl/fl LysMCre +/mice ( Figure S10).
PKM2 is known to phosphorylate STAT3 and regulate the expression of several pro-inflammatory genes. 6,10 We determined PKM2 interaction with STAT3 in neutrophils. Immunoprecipitation showed that PKM2 interacts with STAT3 ( Figure 5D). Immunoblot analysis revealed increased STAT3 phosphorylation in neutrophils isolated from control PKM2 fl/fl Apoe -/mice with stroke compared to mice with sham control (Figure 5E, after 6 hours of reperfusion). On the other hand, we observed significantly reduced STAT3 phosphorylation in neutrophils isolated from PKM2 fl/fl LysMCre +/-Apoe -/mice compared with PKM2 fl/fl Apoe -/mice ( Figure 5E). Because activated STAT3 is known to maintain constitutive NF-κB activity by prolonging NF-κB nuclear retention, 33 we analyzed NF-κB phosphorylation. We found that NF-κB phosphorylation significantly increased in neutrophils isolated from control PKM2 fl/fl Apoe -/mice with stroke compared with mice with sham surgery. PKM2 fl/fl LysMCre +/-Apoe -/mice exhibited reduced NF-κB phosphorylation in neutrophils compared with PKM2 fl/fl Apoe -/mice in the setting of stroke ( Figure 5E). Together, these results suggest that PKM2 regulates post-ischemic inflammation by promoting STAT3 phosphorylation in neutrophils. Next, we determined the effect of PKM2 deletion on glycolytic proton efflux rate (glycoPER) in neutrophils utilizing Seahorse extracellular flux analyzer. We observed that neutrophils from the myeloid specific PKM2 deficient mice exhibit reduced glycoPER at baseline and when stimulated with PMA (100 nM) (Figure S11).

ML265 treatment significantly reduces PKM2 nuclear translocation and neutrophil activation following acute ischemic stroke
To evaluate the therapeutic significance of targeting PKM2 in improving stroke outcome following reperfusion, we used ML265, a small molecule that inhibits PKM2 nuclear translocation by inducing PKM2 tetramerization. 34 We first determined in vivo dose of ML265 that was required to significantly inhibit PKM2 nuclear translocation in neutrophils following stroke. ML265 was administered at the dose of 10, 25, and 50 mg/kg, 5 minutes following reperfusion, and neutrophils were isolated 6 hours post-reperfusion ( Figure 6A). We found that ML265 at the dose of 25 and 50 mg/kg significantly inhibited PKM2 nuclear translocation in neutrophils ( Figure 6B). We, therefore selected a minimal dose of 25 mg/kg for further study and evaluated inflammatory status in the peripheral neutrophils at 6 hours of reperfusion. We observed significantly reduced levels of TNFα, IL-1β, and IL-6 as well as reduced NETosis (P<0.05 versus vehicle, Figure 6CD). Next, we determined whether ML265 inhibits glycolytic rate in neutrophils from the WT mice. We found ML265 (at 10µM and 50 µM) did not reduce glycoPER in PMA (100 ng) activated neutrophils ( Figure S12), suggesting that improved stroke outcome after ML265 treatment is most likely mediated due to the reduced thrombo-inflammation rather than decreased glycolytic rate in neutrophils.

ML265 treated mice exhibited improved long-term sensorimotor outcomes in mice
We assessed stroke outcomes in ML265 treated mice. 10-12 weeks old male WT mice were randomized to receive either ML265 (25 mg/kg) or vehicle, and susceptibility to ischemia/reperfusion injury was evaluated following 60 minutes of ischemia and up to 4 weeks of reperfusion in the filament model ( Figure 7A). The schematic of the study design is shown in Figure S13. Treatments were performed 5 minutes post-reperfusion, and the individuals performing the surgery and behavioral outcomes were blinded to the treatments. A significant reduction in infarct area (≈31%) was observed in ML265 treated mice compared to control-treated mice ( Figure   7B). Next, using the same cohort of mice, we evaluated neurological outcome (mNSS) for up to 4weeks. We found that the ML265 treated group exhibited significantly improved mNSS compared to the vehicle-treated group (Figure 7C). To evaluate the long-term sensorimotor outcome, we performed corner test, rota-rod test, and hanging wire tests. We found that ML265 treated group exhibited significantly improved long-term sensorimotor outcome compared to the vehicle-treated group (Figure 7D-F), while the mortality rate did not differ between the groups ( Figure 7G). Next, we evaluated the effect of targeting PKM2 treatment on stroke outcome in the preexisting comorbid condition of hyperlipidemia. 8-10 weeks old male mice were randomized to receive either ML265 (25 mg/kg) or vehicle, and stroke outcomes were evaluated. We found that ML265 treatment significantly reduced infarct area and improved neurological score in PKM2 fl/fl Apoe -/mice but not in PKM2 fl/fl LysMCre +/-Apoe -/mice ( Figure S14). Comparable stroke outcome in ML265 treated PKM2 fl/fl LysMCre +/-Apoe -/mice and vehicle-treated PKM2 fl/fl LysMCre +/-Apoe -/suggests that, most likely ML265 improves stroke outcomes by inhibiting nuclear PKM2 translocation in myeloid cells.

Discussion
In the past, several strategies have been used to improve stroke outcomes following reperfusion, including neuroprotective, antioxidant, and anti-inflammatory agents. 35 However, most of the strategies have failed in large clinical trials, mainly due to the complexity of human stroke, lack of rigor with the preclinical assessment of these agents, and the design/implementation of a clinical trial. 36 Notably, several preclinical studies used young, healthy mice without preexisting comorbidities. Moreover, with the notable exception of the ESCAPE-1 trial, 37 no phase 3 trial was designed to determine whether neuroprotection could ameliorate the consequences of reperfusion by salvaging penumbra. 38 In the current study, we implemented several STAIR/RIGOR recommendations to overcome methodological shortcomings and demonstrated a mechanistic role for nuclear PKM2 in ischemic stroke pathogenesis. We believe that these findings are novel and may have clinical significance for the following reasons: First, nuclear PKM2 was upregulated in peripheral neutrophils following ischemic stroke in humans and mice, and that contributes to neutrophil hyperactivation. Second, using novel myeloid cell-specific PKM2-deficient mice and utilizing filament and embolic models of stroke, we provide in vivo evidence that PKM2 modulates cerebral ischemia and reperfusion injury in a healthy as well as in a preexisting comorbid condition by regulating thrombo-inflammation. Third, as a translational potential, we demonstrated that therapeutic inhibition of nuclear PKM2 translocation improves stroke outcome and enhances longterm sensorimotor recovery in mice. Our findings provide yet the unidentified role of neutrophil PKM2 in regulating neutrophil hyperactivation and post-stroke thrombo-inflammation.
Inflammation predisposes to ischemic stroke and can trigger several pathogenic aspects that are detrimental to brain salvage and recovery following reperfusion. Gene expression profile changes rapidly in blood after stroke in humans and occurs predominantly in neutrophils. 39 These homeostasis changes in neutrophils, associated with stroke severity, may play an instrumental role by contributing to systemic inflammation. Neutrophils increase stroke severity by several mechanisms that include triggering capillary sludging, generating free radicals, secreting inflammatory mediators, and enhancing thrombosis via the formation of neutrophil-platelet aggregates and NETs. 27,40,41 Herein, we demonstrated that genetic deletion of PKM2 in myeloid cells reduced inflammatory cytokines (TNF-α, IL-1β, and IL-6) and down regulated several proinflammatory genes (MPO, elastase, HIF1, IL-1) within neutrophils following cerebral ischemia/reperfusion. Importantly, we demonstrated that either deletion of PKM2 in myeloid cells or inhibition of nuclear PKM2 translocation with small molecule ML265 improved short and longterm functional outcome. IL-6 in stroke patients is known to be associated with poor clinical outcomes. 42 TNF-α and IL-1β are known to enhance leukocyte migration to the ischemic region, promote necrosis, increase endothelial dysfunction, disrupt BBB and increase edema formation following stroke. 43 Together, these observations suggest that nuclear PKM2 in neutrophils drives post-ischemic inflammatory response by upregulating several pro-inflammatory genes and thereby exacerbates stroke outcome.
Following a stroke, the ischemic core undergoes permanent necrotic cell death while the salvageable penumbral tissue is prone to further neuronal cell death due to multiple mechanisms that include excitotoxicity, oxidative stress, ionic imbalance, and inflammation. 44 Although intravenous thrombolysis and mechanical thrombectomy are the pillars of acute stroke care, they do not target the mechanisms that can cause secondary brain damage in the penumbral tissue.
Moreover, the "no-reflow" phenomenon, which is characterized by secondary microvascular cerebral thrombosis (primarily platelets and neutrophils) and penumbral tissue damage despite successful recanalization, still remains a unique problem. Herein, we observed that PKM2 regulates NETs formation in the setting of ischemic stroke and myeloid cell-specific deficiency of PKM2 improved regional cerebral blood flow following reperfusion. Collectively, these observations suggest that PKM2 may potentiate post-ischemic secondary thrombosis, by promoting NET formation, in addition to inflammation. NETs, which are composed of chromatin and antimicrobial proteins (histones, myeloperoxidase, and elastase), are known to contribute to stroke pathogenesis. 25,27,45 Indeed, we found reduced expression of MPO and elastase in peripheral neutrophils of myeloid-specific PKM2 deficient mice following stroke. The precise mechanism by which PKM2 potentiates NETs formation remains unclear and remains an area of investigation.
However, we speculate that nuclear PKM2 may promote NETosis by aggravating pro-inflammatory environment.
We also investigated the molecular mechanism by which myeloid cell specific PKM2 promotes stroke exacerbation. Unlike other isoforms of PK, that exist and function as stable tetramers, PKM2 subunits forms tetramers and dimers composed of the same monomers but with different biological activities. Evidence suggests that only dimeric PKM2 can enter nucleus to exert protein kinase activity. Nuclear PKM2 interacts with STAT3 and enhances its phosphorylation at Y705 and is known to contributing to cell proliferation in the cancer cell 15 and inflammatory cytokine production in macrophages. 19 Although it is known that PKM2 interacts with STAT3 in other cell types, the role of PKM2 in STAT3 phosphorylation in peripheral neutrophils in the context of stroke is not defined. In the current study, we observed that similar to other cell types, PKM2 interacts with STAT3 in neutrophils. Genetic deletion of PKM2 significantly reduced phosphorylation of STAT3 levels in peripheral neutrophils following stroke. STAT3 regulates G-CSF-dependent accumulation of immature bone marrow neutrophils and acute G-CSF-induced neutrophil mobilization, 46 indicating the key role of STAT3 in neutrophil function during a proinflammatory environment. Additionally, STAT3-deficient neutrophils have a cell-autonomous defect in migration toward ligands for CXCR2, as well as defective MPO secretion 47 . We found reduced MPO and elastase levels after stroke in PKM2 deficient neutrophils in line with these observations. Crosstalk between STAT3 and NF-κB has been reported by several studies, including activation of STAT3 by NF-κB regulated factors such as IL-6. 33,48 Recently, it was shown that activated STAT3 maintains constitutive NF-κB activity by prolonging NF-κB nuclear retention 33 .
Consistent with these observations, we found that PKM2 deficiency in peripheral neutrophils results in reduced NF-κB phosphorylation, reduced NF-κB activity, and decreased pro-inflammatory cytokine production after ischemic stroke. Although neutrophils from myeloid specific PKM2 deficient mice exhibited reduced glycolytic rate, neutrophils after ML265 treatment did not exhibit reduced glycolysis, suggesting that most likely the PKM2-STAT3-NF-κB axis regulates neutrophil hyperactivation following cerebral ischemia and thereby contributes to stroke severity.
The strength of the current study is that we determined the role of PKM2 using both genetic and pharmacological approaches in two different stroke models, in both sexes and in mice with comorbidities. Despite strengths, our study has limitations. First, PKM2 is expressed by other cell types, including endothelial cells, monocytes/macrophages, T cells, and platelets. The possibility of potential unexpected and adverse physiological side effects of blocking nuclear PKM2 in other cell types cannot be ruled out. Nevertheless, we speculate that such a scenario is unlikely because of the acute nature and single-dose treatment. Second, previous studies by other groups have suggested an important role of macrophage 49 and T cells in stroke pathogenesis. 50 Thus, the possibility of macrophage or T cell derived PKM2 in mediating stroke outcome cannot be completely ruled out.
Extending these studies to other species, for example, hypertensive rats may further validate therapeutic potential of these novel findings. In summary, we have demonstrated a mechanistic role of PKM2 in regulating neutrophil hyperactivation and acute ischemic stroke.

Declaration of Interest
The authors declare that they have no competing interests.    climbing, body proprioception, and responses to vibrissae touch (higher score indicates a better outcome). Sensorimotor recovery in the same mice as analyzed by asymmetry index in cylinder test (D), fall latency in accelerated rota-rod test (E), motor strength in hanging-wire test (F), and right turn ratio in corner test (G). The animals that successfully completed the particular neurological test were included in the analysis (see exclusion/inclusion criteria in methods). Data are mean ± SEM (B) and median ± range (C, D, E, F, and G). n=10 (male mice). Statistical analysis: unpaired t-test (B), Two-Way Repeated Measures ANOVA (Kruskal-Wallis test) followed by Fisher's LSD test (D, E, F, and G). . Right: Thrombotic index as defined by the ratio of occluded brain vessels to the total brain vessels in the ipsilateral hemisphere (C). Brain homogenates from the infarcted and peri-infarcted area following 1-hour ischemia/23 hours reperfusion were processed for Western blotting: Representative Western blots and densitometric analysis of fibrin(ogen) and platelets (CD4-positive). β-Actin was used as a loading control. All data are from male mice and are mean ± SEM. n=5(A), n=4 (B and C). Statistical analysis: Two-Way Repeated Measures ANOVA (Kruskal-Wallis test) followed by Fisher's LSD test (A), unpaired t-test (B and C).  The quantitative data of cytosolic and nuclear PKM2 intensity (normalized to the intensity LaminB1/GAPDH at each time point) are shown on the right. (C) TNF-α, IL-1β, and IL-6 levels in neutrophils isolated 6 hours post-reperfusion from each group as analyzed by ELISA. TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin 1 beta; IL6, interleukin 6. (D) Immunofluorescence analysis of NETs from the neutrophils isolated 6 hours post-reperfusion. Neutrophils were stimulated with the suboptimal concentration of PMA (10 ng/mL), and NETs were visualized by SYTOX Green stain. Scale bar 100 µM. Quantification is shown on the right. PMA, phorbol 12myristate 13-acetate. Data are from male WT mice and are mean ± SEM, n=3 (B), n=4 (C) and n=5 (D). Statistical analysis: Two-Way Repeated Measures ANOVA (Kruskal-Wallis test) followed by Fisher's LSD test (B), unpaired t-test (C and D).