tPA Mobilizes Immune Cells that Exacerbate Hemorrhagic Transformation in Stroke.

Rationale: Hemorrhagic complications represent a major limitation of intravenous thrombolysis using tissue plasminogen activator (tPA) in patients with ischemic stroke. The expression of tPA receptors on immune cells raises the question of what effects tPA exerts on these cells and whether these effects contribute to thrombolysis-related hemorrhagic transformation. Objective: We aim to determine the impact of tPA on immune cells and investigate the association between observed immune alteration with hemorrhagic transformation in ischemic stroke patients and in a rat model of embolic stroke. Methods and Results: Paired blood samples were collected before and 1 hour after tPA infusion from 71 ischemic stroke patients. Control blood samples were collected from 27 ischemic stroke patients without tPA treatment. A rat embolic middle cerebral artery occlusion model was adopted to investigate the underlying mechanisms of hemorrhagic transformation. We report that tPA induces a swift surge of circulating neutrophils and T cells with profoundly altered molecular features in ischemic stroke patients and a rat model of focal embolic stroke. tPA exacerbates endothelial injury, increases adhesion and migration of neutrophils and T cells, which are associated with brain hemorrhage in rats subjected to embolic stroke. Genetic ablation of annexin A2 in neutrophils and T cells diminishes the effect of tPA on these cells. Decoupling the interaction between mobilized neutrophils/T cells and the neurovascular unit, achieved via a sphingosine-1-phosphate receptor 1 modulator RP101075 and a CCL2 synthesis inhibitor bindarit, which block lymphocyte egress and myeloid cell recruitment, respectively, attenuates hemorrhagic transformation and improves neurological function after tPA thrombolysis. Conclusions: Our findings suggest that immune invasion of the neurovascular unit represents a previously unrecognized mechanism underlying tPA-mediated brain hemorrhage, which can be overcome by precise immune modulation during thrombolytic therapy.

HT occurs when blood products extravasate into the infarct area during reperfusion. This is caused by increased permeability of blood-brain barrier (BBB) and vascular basal lamina dysfunction. tPA-mediated thrombolysis increases the risk of HT. Recent research suggests that the nonthrombolytic effects of tPA may contribute to this devastating complication. tPA binds several receptors, including annexin A2, LRP (low-density lipoprotein receptor-related protein) 1, and the NMDAR (N-methyl-D-aspartate receptor), resulting in differential downstream biological effects. [3][4][5][6][7] For instance, tPA compromises BBB integrity via LRP1 expressed on endothelial cells, microglia, and astrocytic endfeet. 5,8,9 tPA also activates PDGF-CC (platelet-derived growth factor-CC) 10 and kallikrein, 11 which promote BBB disruption.
The presence of tPA receptors on immune cells prompts the question of what effects tPA exerts on these cells and whether these effects contribute to HT. Indeed, tPA alters the activation status of monocytes/ macrophages exposed to lipopolysaccharide 6 and also increases leukocyte infiltration into lung, kidney, and cremaster muscle during ischemia/reperfusion. [12][13][14][15] Among leukocyte subsets, neutrophils have been linked to intracerebral hemorrhage after thrombolysis in animal models 16,17 and patients with ischemic stroke 18,19 ; however, several key questions remain unanswered. First, as all the published studies evaluate circulating immune response at 24 hours after tPA thrombolysis, at that time HT has already occurred, it is unknown whether the augmented circulating leukocyte response is a cause Nonstandard Abbreviations and Acronyms BBB blood-brain barrier CCL2 C-C motif chemokine ligand 2 CCR2 C-C motif chemokine receptor 2 eMCAO embolic middle cerebral artery occlusion HT hemorrhagic transformation IL interleukin LRP low-density lipoprotein receptor-related protein MAPK mitogen-activated protein kinase MMP matrix metallopeptidase NMDAR N-methyl-D-aspartate receptor PDGF-CC platelet-derived growth factor-CC S1PR sphingosine-1-phosphate receptor tPA tissue-type plasminogen activator

Novelty and Significance
What Is Known?
• tPA (tissue-type plasminogen activator) is the only Food and Drug Administration-approved pharmacological treatment for acute ischemic stroke. The use of tPA is limited by its narrow time window and a risk of intracerebral hemorrhage. • In addition to thrombolysis, tPA has pleiotropic actions that may contribute to hemorrhagic complications.

What New Information Does This Article
Contribute?
• In patients with ischemic stroke and a rat model of embolic stroke, tPA swiftly induces a surge of circulating immune cells. • tPA profoundly alters transcriptomic profile including a significant increase of genes related to chemotaxis, activation, and infiltration in neutrophils. • tPA mobilizes peripheral neutrophils and T cells via annexin A2. • Decoupling interaction between neutrophils/T cells and neurovascular unit reduces hemorrhagic transformation and improves neurological outcome following tPA thrombolysis.
Acute ischemic stroke is a serious health problem worldwide. Current therapies for this devastating disease are limited. tPA administered within 4.5 hours of onset promotes neurological recovery of stroke patients, in part, by opening up the clotted blood vessels. However, the narrow time frame and the risk of intracerebral hemorrhage after tPA treatment pose major drawbacks to its clinical application. Peripheral immune cells infiltrating into ischemic brain exacerbate blood-brain barrier disruption and neurovascular injury. However, it remains unknown what effects tPA has on its receptor-bearing immune cells and whether these effects contribute to tPA-related hemorrhagic transformation. In this study, we have demonstrated that tPA swiftly mobilizes circulating neutrophils and T cells in patients with acute ischemic stroke and a rat model of embolic stroke. These neutrophils and T cells home to the ischemic brain and contribute to the emergence of hemorrhagic transformation.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

RESULTS tPA Swiftly Mobilizes Neutrophils and T Cells in Ischemic Stroke Patients and in a Rat Model of Focal Embolic Stroke
To investigate how tPA influences the immune system, we analyzed circulating myeloid cells and lymphocytes from 71 ischemic stroke patients who received tPA and 27 without tPA. Total leukocyte and lymphocyte counts were increased by ≈15% and 19%, respectively, as early as 1 hour after tPA administration compared with cell counts before tPA treatment and control patients (Figure I in the Data Supplement). Further analysis of cellular subsets via flow cytometry revealed that neutrophils increased by 31%; T cells, including cluster of differentiation (CD) 4 T cells increased by 20%, and CD8 + T cells increased by 26% (Figure 1). Neutrophils and T cells were the main cell types influenced by tPA administration, whereas counts of monocytes, B cells, and natural killer cells remained relatively stable before and after tPA treatment; these cell counts were also comparable to those of control patients ( Figure II in the Data Supplement). Next, we induced embolic stroke in rats by delivering a 4-cm fibrin-enriched clot to the origin of middle cerebral artery via a catheter and administered tPA at 3 hours post-ischemia ( Figure III in the Data Supplement). This model more accurately recapitulates the key components of thromboembolic stroke and subsequent tPAinduced thrombolysis in human ischemic stroke, relative to the intraluminal filament middle cerebral artery occlusion model. 21 In eMCAO rats, the count of circulating neutrophils was increased at 1 hour after tPA administration by ≈4-fold as compared with controls receiving saline. The increase of circulating neutrophils was sustained up to 12 hours post-thrombolysis (Figure 2A and 2B). CD4 + and CD8 + T cells were also increased by ≈2fold in peripheral blood samples of eMCAO rats treated with tPA, with elevation of CD8 + T cells detected as early as 15 minutes after tPA administration ( Figure 2B). A transient increase in B-cell count was also observed following tPA treatment ( Figure  Notably, cell counts in peripheral blood samples obtained from sham rats receiving tPA did not exhibit significant changes. The reason for this disparity is unclear but could imply that brain ischemia renders immune cells more susceptible to tPA. To further characterize the newly mobilized neutrophils after tPA thrombolysis, we sorted circulating neutrophils from rats and utilized RNA sequencing to characterize the molecular features. In circulating neutrophils from eMCAO rats receiving tPA, we identified a total of 2575 altered genes (upregulated, 1449 genes; downregulated, 1126 genes) after tPA thrombolysis as compared with eMCAO group receiving PBS. Moreover, 820 altered genes were identified in eMCAO+PBS group versus eMCAO+tPA group (Figure VIA and VIB in the Data Supplement). Among the altered genes, we found a significant increase of genes related to neutrophil chemotaxis, activation, and infiltration, including CCR2 (C-C motif chemokine receptor 2) and MMP (matrix metallopeptidase) 9 ( Figure 2C and 2D). This result indicates that tPA thrombolysis induces profound changes in peripheral neutrophils of eMCAO rats. Similarly, the expression of the adhesion molecule receptor CD49d and activation molecule CD69 was increased in CD4 + and CD8 + T cells following tPA administration ( Figure 2E). Together, these results indicate that tPA preferentially augments peripheral neutrophils and T cells in ischemic stroke patients and in rats following eMCAO.

Neutrophil and T-Cell Invasion of the Cerebrovascular Compartment Is Associated With HT Following tPA Thrombolysis
We next examined the destination of migrating immune cells following tPA thrombolysis in eMCAO rats. Compared with saline-treated rats, neutrophils, CD4 + T cells, and CD8 + T cells were observed in ischemic brain 4 hours post-tPA administration, becoming more pronounced at 12 hours ( Figure 3A). The counts of braininfiltrating neutrophils and T cells were associated with their corresponding counts in blood after tPA XCthrombolysis ( Figure VII in the Data Supplement). In addition, upregulation of CCR2, CXCR2 (C-X-C motif chemokine receptor 2), MMP9, and TLR4 (toll-like receptor 4) was observed in brain-infiltrating neutrophils ( Figure VIII in the Data Supplement). Importantly, while the infiltration of these cells in the brain parenchyma of eMCAO rats was not observed at 1 hour after tPA administration, the accumulation of neutrophils and T cells in the microvessel lumen of the ipsilateral hemisphere was evident at this early time ( Figure 3B and 3C).
Having determined that these activated immune cells were physically associated with the cerebral blood vessel endothelium, we went on to test whether this finding is related to postthrombolysis HT. To this end, we analyzed the relationship between counts of circulating cells at 1 hour after tPA infusion and cerebral hemorrhage volume at 24 hours after eMCAO. Intracerebral hemorrhage was measured by 7T rodent magnetic resonance imaging scanning using a T2* sequence, which is sensitive to brain hemorrhage. 22 Linear regression analysis confirmed that brain hemorrhage volume was associated with the numbers of neutrophils, CD4 + T cells, and CD8 + T cells in peripheral blood samples at 1 hour after tPA infusion ( Figure 3D). Considering that HT occurs at a median of 5 to 10 hours following tPA administration, these data suggest that tPA-mobilized neutrophils and T cells may contribute to the development of brain hemorrhage.

Direct Effects of tPA on Neutrophils and T Cells and Their Impact on Endothelial Injury Following Hypoxia and Glucose Deprivation
To determine whether tPA directly acts on neutrophils and T cells, we sorted these cells from blood samples obtained from rats at 3 hours post-eMCAO and sham controls. Following exposure to 1 to 100 µg/mL tPA, flow cytometry analysis was performed to evaluate the

tPA (tissue-type plasminogen activator) induces a surge of circulating neutrophils and T cells in patients with ischemic stroke.
Blood leukocytes were isolated from 71 patients with ischemic stroke before and 1 h after receiving tPA thrombolysis and 27 patients without tPA treatment (controls) at ≈4 and ≈7 h post-onset. Peripheral immune cell subsets were analyzed by flow cytometry. Demographic information and the baseline characteristics of the participating subjects are provided in Table I in the Data Supplement. A, Flow cytometry dot plots from representative subjects show an elevation of neutrophils (CD16 + CD14 − ) in the peripheral blood of ischemic stroke patients receiving tPA thrombolysis; numbers in the gates indicate percentage of neutrophils of total leukocytes. B, Representative dot plots show the elevation of CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ) in blood of patients received tPA thrombolysis. Numbers indicate the percentage of CD4 + T cells or CD8 + T cells in total lymphocytes. C-E, Absolute numbers of neutrophils, CD4 + T cells, and CD8 + T cells from individual subject and statistical analysis; data are shown as mean±SEM. Wilcoxon signed-rank test was used to compare cell numbers before and after tPA administration in patients with stroke. Mann-Whitney U test was used to compare the cell numbers in stroke patients receiving tPA and stroke patients without tPA. *P<0.05, **P<0.01. CD indicates cluster of differentiation. Circulation Research. 2021;128:62-75. DOI: 10.1161/CIRCRESAHA.120.317596 expression of adhesion molecules in these cells. A timeand dose-dependent elevation of CCR2 on neutrophils and CD49d on T cells was observed at 12 hours in cells isolated from eMCAO rats; no significant upregulation in cells from sham rats was detected ( Figure 4A through 4C). Neutrophils exhibited a quicker response to tPA treatment than did T cells, as CCR2 expression level was increased as early as 1 hour after tPA treatment at 100 µg/mL ( Figure 4A), while the CD49d expression of T cells was not upregulated until 12 hours after tPA treatment ( Figure 4B and 4C). To compare newly released versus old neutrophils and T cells, we injected Sulfo-NHS-LC Biotin into recipient rats to label circulating neutrophils and T cells. 23 Such an approach allows identification of freshly released neutrophils and T cells after 1 hour as Biotin − , whereas old neutrophils and T cells are Biotin + . In eMCAO rats receiving Sulfo-NHS-LC-Biotin injection, we found upregulation of CCR2 and MMP9

tPA (tissue-type plasminogen activator) swiftly mobilizes peripheral neutrophils and T cells after embolic stroke in rats.
Three-month male Wistar rats were subjected to embolic middle cerebral artery occlusion (eMCAO); tPA was given 3 h after surgery. Blood samples were collected from the internal iliac vein beginning before surgery up to 12 h after tPA thrombolysis to confirm the findings observed in patients and to delineate the dynamic changes of cell populations. Cell numbers and their expression of adhesion molecular receptors were detected using flow cytometry. A, Flow cytometry dot plots show the gating strategy of neutrophil (CD11b + granulocyte + ), CD4 + T cell (CD3 + CD4 + ), and CD8 + T cell (CD3 + CD4 + ) in rat blood. B, The dynamics of absolute cell numbers of circulating neutrophils, CD4 + cells, and CD8 + T cells in sham and eMCAO rats treated with saline or tPA from baseline to 12 h post-tPA administration. n=5 to 8 in each group. C, Circulating neutrophils were sorted from sham rats and eMCAO rats at 12 h after tPA thrombolysis. Unbiased RNA sequencing was used to characterize the transcriptional alterations related to tPA-induced neutrophil mobilization. Heat map shows the Z score of top 20 upregulated genes related to neutrophil chemotaxis, activation, and infiltration. Higher Z score indicates upregulation of genes in red; downregulated genes with a negative Z score are in blue. Dendrogram depicts the similarity among genes corresponding to their expression level. n=3 in each group. D, Flow cytometry analysis revealed activity of circulating neutrophils in indicated groups at 12 h after tPA thrombolysis; n=6 in each group. E, Flow cytometry analysis of CD49d and CD69 expression in circulating CD4 + and CD8 + T cells at 12 h after tPA infusion; n=6 in each group. Data are shown as mean±SEM; comparison was conducted by 2-way ANOVA (B, D, and E). CD indicates cluster of differentiation; CXCR2, C-X-C motif chemokine receptor 2; FSC: forward scatter; SSC, side scatter; and TLR4, toll-like receptor 4.

Figure 3. tPA (tissue-type plasminogen activator) facilitates the migration of peripheral neutrophils and T cells to the neurovascular unit that was associated with hemorrhagic transformation.
A, Brain tissue of embolic middle cerebral artery occlusion (eMCAO) rats receiving saline or tPA treatment at 3 h after surgery was analyzed using flow cytometry to determine the brain infiltration of neutrophils and T cells. Dot plots at the top show the gating of neutrophils (CD45 + CD11b + granulocyte + ), CD4 + T cells (CD45 + CD3 + CD4 + ), and CD8 + T cells (CD45 + CD3 + CD8 + ) of rat brain tissue after eMCAO. Statistical graphs at the bottom show the absolute cell numbers of neutrophils, CD4 + T cells, and CD8 + T cells in the brain tissue of eMCAO rats at indicated time points after tPA administration. n=6 in each group. B and C, Left, Immunohistochemical staining of brain slices of eMCAO rats at 1 and 12 h after tPA or saline administration for CD3 (B), neutrophil (C), and CD31 to detect the immune cells that accumulated within the microvessel lumen, as well as which infiltrated into the parenchyma that was located within the ipsilateral ischemic area. White triangles signify cells accumulated in the microvessel lumen, white stars denote the cells infiltrating into the parenchyma. Scale bar=50 µm. Right, Graphs show the absolute cell numbers of T cells (B) and neutrophils (C) within blood vessels or parenchyma and statistical analysis. n=10 in each group. D, Brain hemorrhage volume of eMCAO rats treated by tPA was measured by magnetic resonance imaging T2* images at 24 h after ischemia. E-G, The correlation between brain hemorrhage volume at 24 h after stroke and circulatory cell counts of neutrophils, CD4 + T cells, and CD8 + T cells at 1 h after tPA thrombolysis, n=12. Data are shown as mean±SEM; 2-way ANOVA was performed in A-C and t test in D. Linear regression analysis was performed in E-G. Linear regression line, R 2 (goodness of fit), and P are shown in the graphs. *P<0.05, **P<0.01. CD indicates cluster of differentiation; FSC, forward scatter; and SSC, side scatter. in newly released Biotin − neutrophils versus old Biotin + neutrophils ( Figure IX in the Data Supplement). Similarly, we observed upregulation of CD69 and CD49d in newly released Biotin − T cells versus old Biotin + T cells ( Figure  IX in the Data Supplement). As tPA can induce endothelial injury, we also determined whether tPA-induced endothelial injury could contribute to immune activation after tPA administration. For this purpose, we performed coculture experiments using endothelial cells exposed to tPA and immune cells isolated from eMCAO rats. We found that tPA-induced endothelial injury alone is insufficient to activate neutrophils and T cells ( Figure X in the Data Supplement).
Next, we sought to determine whether tPA-activated neutrophils or T cells exacerbate endothelial injury-the key pathological mechanism underlying HT 24 . An in vitro BBB model was established by seeding a monolayer of endothelial bEND3 cells on collagen and fibronectin-coated inserts in a transwell culture system. This in vitro BBB model was exposed to hypoxia combined with . Neutrophils and T cells exposed to tPA (tissue-type plasminogen activator) exacerbate endothelial cell injury after hypoxia and glucose deprivation (HGD). A-C, Isolated neutrophils and T cells from embolic middle cerebral artery occlusion (eMCAO) or sham rats at 3 h after surgery were cultured with tPA at indicated concentrations. Flow cytometry analysis of CCR2 (C-C motif chemokine receptor 2) expression in neutrophils (A) and CD49d in CD4 + T cells (B) and CD8 + T cells (C) at 12 h after tPA exposure. n=6 per group. D-F, Endothelial bEND3 cells seeded in cell culture inserts (D) or cover slides (E) coated by collagen and fibronectin, or in plates (F), were exposed to 4 h of HGD. After this, cells were cocultured with neutrophils or T cells treated with or without tPA. For neutrophils, tPA was pretreated for 4 h because of its quick response. For T cells, tPA was pretreated for 12 h. Neutrophils and T cells were washed with PBS to remove tPA in the medium before coculture. The ratio of endothelial cells to neutrophils/T cells was 2:1. D, Blood-brain barrier permeability was determined by measuring the FITC (Fluorescein)-dextran diffused from the upper chamber to lower chamber over 12 h. n=6 in each group. E, Immunofluorescence staining images show the tight junction protein claudin-5 expression of endothelial cells. Scale bar=50 µm. F, Twelve hours after coculture, cell lysates were collected for quantitative measurement of claudin-5 by Western blot. n=6 per group. Data are shown as mean±SEM; 2-way ANOVA in A-D or 1-way ANOVA in F. *P<0.05, **P<0.01. CD indicates cluster of differentiation; and FITC, Fluorescein. glucose deprivation for 4 hours, followed by coculture with tPA-treated neutrophils or T cells. FITC (Fluorescein)dextran that leaked from the upper chamber to the lower chamber was quantified to assess the permeability. Relative to baseline, hypoxia glucose deprivation increased the diffusion of FITC-dextran as early as 1 hour after hypoxia induction. tPA-treated neutrophils or T cells exacerbated hypoxia glucose deprivation-induced FITCdextran diffusion by ≈2-fold, whereas untreated neutrophils or T cells produced only a trend toward increasing BBB leakage without statistical significance ( Figure 4D). In addition, tPA-treated neutrophils or T cells promoted the degradation of the tight junction protein claudin-5 ( Figure 4E and 4F). These findings suggest that tPA directly activates neutrophils and T cells, which exacerbates BBB disruption after tPA thrombolysis.

Annexin A2 Bridges tPA and Immune Cells After Thrombolysis
To identify the tPA receptor that mediates the effects of tPA on immune cells, we screened the expression of several tPA receptors (LRP1, LRP4, annexin A2, and NMDAR) in immune cells collected from blood of eMCAO rats. Among examined receptors, annexin A2 was highly expressed by ≈40% of neutrophils. Comparison of annexin A2 expression among different immune cell types revealed that neutrophils and T cells were the predominant cell populations expressing annexin A2 ( Figure XI in the Data Supplement). We then examined the potential downstream pathways that mediate the effects of tPA on immune cells. KEGG pathway enrichment analysis revealed that MAPK (mitogen-activated protein kinase) signaling pathway was the most enriched pathway in neutrophils sorted from eMCAO rats following tPA thrombolysis; genes of MAPK family members are highly expressed in neutrophils of eMCAO rats receiving tPA ( Figure 5A and 5B), suggesting that MAPK pathway might mediate the activation of immune cells after tPA administration. Western blot analysis demonstrates activated p38 MAPK in neutrophils of eMCAO rats after tPA treatment ( Figure 5C). In addition, a selective p38 MAPK inhibitor prevented the activation of neutrophils by tPA in eMCAO rats ( Figure 5D), suggesting the contribution of tPA-annexin 2-MAPK axis to tPA-induced mobilization of immune cells.
Next, we tested whether annexin A2 is necessary for the effect of tPA on neutrophils or T cells. Neutrophils and T cells isolated from healthy rat bone marrow or spleen, respectively, were transfected with annexin A2 siRNA to knock down annexin A2 expression ( Figure XII in the Data Supplement). After labeling with Molday ION Rhodamine B, 5×10 6 cells were injected via tail vein to recipient rats before eMCAO surgery and tPA administration ( Figure 6A). Molday ION Rhodamine B is a fluorescence-conjugated nanoparticle that can be used to trace extrinsically transferred cells in vivo as we reported previously. 25 Compared with cells transfected with control siRNA, annexin A2 knockdown inhibited tPA-induced activation of p38 MAPK ( Figure XIII in the Data Supplement). The expression of CCR2 and CD49d on neutrophils and T cells was also reduced after annexin A2 knockdown at 12 hours post-tPA administration (Figure 6D, 6E, and 6G through 6J), together with reduced transmigration of these cells into the ischemic brain ( Figure 6F and 6K). These findings suggest that the tPA receptor annexin A2 is required for the effects of tPA on peripheral immune cells.

Suppression of the Transmigration of Neutrophils and T Cells Reduced tPA-Associated HT and Improved Neurological Function Following eMCAO
We next sought to examine whether inhibition of T cell or neutrophil migration reduces the extent of HT associated with tPA thrombolysis. To inhibit tPA-induced immune activation, an anti-annexin A2 mAb was given to eMCAO rats immediately before tPA administration. We found significant reduction in brain hemorrhage volume in eMCAO rats receiving anti-annexin A2 mAb ( Figure XIV in the Data Supplement). We then tested 2 immune modulating drugs that have translational potential. RP101075 is a second generation of S1PR (sphingosine-1-phosphate receptor) modulator, which selectively inhibits S1PR1-dependent lymphocytes egress from secondary lymphoid organs. 26 Compared with eMCAO rats receiving saline, eMCAO rats receiving tPA at 3 hours after ischemia had significantly increased brain hemorrhage at 24 hours poststroke (saline versus tPA, 5.6±0.7 versus 14.7±1.4 mm 3 ) without amelioration of infarction ( Figure 7A through 7F). In eMCAO rats receiving RP101075, we found significantly reduced numbers of circulating and brain-infiltrating T cells ( Figure XVA and XVB in the Data Supplement). RP101075 significantly reduced brain hemorrhage in eMCAO rats receiving tPA with a 46% reduction as compared with tPA-treated rats (tPA+RP101075 versus tPA, 7.9±1.5 versus 14.7±1.4 mm 3 ). In addition, the infarct volume of tPA+RP101075treated eMCAO rats was 40% smaller than that of tPAtreated rats (tPA+RP101075 versus tPA, 138.9±12.7 versus 231.4±14.7 mm 3 ). Although tPA thrombolysis at 3 hours after eMCAO did not significantly improve the neurological deficit as compared with eMCAO rats receiving saline, the combination of RP101075 and tPA significantly reduced neurological deficits, neuronal death, and improved long-term sensorimotor function up to 4 weeks after eMCAO (Figure 7G through 7I; Figure  XVI in the Data Supplement).
In addition, we examined whether inhibition of neutrophil migration using bindarit reduces tPA-associated hemorrhage. Bindarit is a CCL2 (C-C motif chemokine ligand 2) inhibitor that blocks the migration of myeloid cells including neutrophils. 27 Importantly, tPA administration upregulated CCR2 expression on neutrophils, suggesting the involvement of CCL2-CCR2 pathway in tPA-mediated neutrophil transmigration. The extent of neutrophil infiltration into brain parenchyma was reduced in bindarit-treated rats ( Figure XVC and XVD in the Data Supplement). Combination of bindarit with tPA reduced volume of brain hemorrhage by 34% compared with tPA-treated rats at 24 hours post-ischemia (tPA+bindarit versus tPA, 9.7±1.5 versus 14.7±1.4 mm 3 ). Infarct volume was reduced by 36% in rats receiving combined treatment of tPA and bindarit relative to rats receiving tPA alone (tPA+bindarit versus tPA, 146.2±19.0 versus 231.4±14.7 mm 3 ). Bindarit also reduced acute neurological deficits at 24 hours after ischemia and improved sensorimotor function at 4 weeks ( Figure 7G through 7I). These data as in whole suggest that inhibition of neutrophil and lymphocyte transmigration may reduce the hemorrhagic risk and improve the efficacy of tPA thrombolysis for ischemic stroke.

DISCUSSION
Previous studies suggest that tPA upregulates the expression of MMPs on brain vascular endothelium 8 and accelerates the degradation of the extracellular matrix of blood vessels, thereby contributing to HT following thrombolytic therapy. In addition to directly activating A and B, Circulating neutrophils were sorted from rats of indicated groups at 12 h post-tPA thrombolysis. RNA sequencing was performed to characterize gene profiles of neutrophils. A, KEGG pathway enrichment analysis shows the top 20 signaling pathways that are enriched in the altered genes between eMCAO+saline group and eMCAO+tPA group. B, Heat map shows the Z score of the top 20 upregulated genes related to MAPK pathway. Upregulated genes are in red. Downregulated genes are in blue. Dendrogram depicts the hierarchical relationship among genes related to the MAPK pathway, n=3 per group. C, Western blot detection of phosphorylated p38 MAPK (p-p38) and p38 MAPK (p38) of neutrophils sorted from peripheral blood of rats of indicated groups. n=6 per group. D, A selective p38 MAPK inhibitor VX-702 was given to eMCAO rats at a dose of 10 mg/kg, immediately before tPA administration. eMCAO rats receiving saline were used as control. The expression of CCR2 (C-C motif chemokine receptor 2) and MMP (matrix metallopeptidase) 9 in neutrophils was measured using flow cytometry at 12 h after tPA administration. n=8 per group. Data are shown as mean±SEM; 1-way ANOVA. *P<0.05, **P<0.01. AMPK,5' indicates adenosine monophosphate-activated protein kinase; mTOR, mammalian target of rapamycin; Rap1, Ras-related protein 1; TNF, tumor necrosis factor; and VEGF, vascular endothelial growth factor. endothelial cells, the present study demonstrates that tPA mobilizes peripheral neutrophils and T cells, which transmigrate to the brain vasculature. Neutrophils and T cells exposed to tPA subsequently exacerbate BBB disruption and promote intracerebral hemorrhage. Further, we show that the action of tPA on neutrophils and T cells requires annexin A2 and involves the downstream MAPK pathway. These results suggest that tPA-mediated neurovascular inflammation represents a new mechanism underlying HT following thrombolytic therapy in ischemic stroke ( Figure XVII in the Data Supplement).
The rationale of studying the action of tPA on immune cells as early as 1 hour after tPA administration is manifold. First, tPA is quickly metabolized in human plasma with a half-life of ≈5 minutes. Hence, any significant effects directly related to tPA administration would occur soon after infusion. Second, HT is the major adverse event related to tPA administration and mostly occurs within 24 hours of treatment, with a median onset from 5 to 10 hours. 2 Consequently, any discernible effects at later than 24 hours is presumed to be secondary effects to HT because the direct effects of tPA would have subsided by this time. Therefore, 24 hours has commonly been used as the time-point to assess the effects of tPA and related adverse events. 18,28,29 In the present study, neutrophil and T-cell populations were swiftly increased in peripheral blood samples obtained from ischemic stroke patients as early as 1 hour post-tPA infusion, implying that the action of tPA on the peripheral immune system occurred before the emergence of adverse events within the brain.
In some stroke cases, HT occurs as a pathological consequence of brain infarction. Particularly following large and embolic stroke, 24 this rate is worsened by intravenous tPA administration. A meta-analysis of 12 trials demonstrates that intravenous tPA causes 60 additional symptomatic intracerebral hemorrhages per 1000 treated stroke patients. 30 Postthrombolysis hemorrhage is the most feared complication of intravenous thrombolysis, with almost 50% mortality in cases with symptomatic hemorrhage following tPA. 24 HT occurs when the integrity of the endothelial cells lining brain vasculature and BBB becomes compromised. 24 Endogenous tPA engendered during brain ischemia also increases the permeability of BBB, which may prime microvessels for the deleterious extravascular effects of therapeutically administered exogenous tPA. 5,8,10 Increased BBB permeability before tPA treatment, which is determined by stroke severity and time to treatment, is thought to promote leakage of Groups of embolic middle cerebral artery occlusion (eMCAO) rats received saline or tPA at 3 h after surgery. The S1PR (sphingosine-1phosphate receptor) 1 modulator RP101075 (0.6 mg/kg) or CCL2 (C-C motif chemokine ligand 2) inhibitor bindarit (50 mg/kg) was given by oral gavage immediately before tPA administration. A and B, At 24 h after eMCAO, infarct volume and intracerebral hemorrhage volume were evaluated by magnetic resonance imaging (MRI) scanning. T2-weighted images (T2WI) of rat brains show the ischemic lesion (red dashed line; A) and hemorrhagic lesions (blue arrow; B). C and D, Gross brain images (C) and histology staining images (D) show the brain hemorrhage in the ipsilateral hemisphere of eMCAO rats of indicated groups at 24 h after ischemia. Scale bar=50 µm. E and F, Measurement of infarct volume (E) and hemorrhage volume (F) based on MRI images of eMCAO rats in indicated groups. G, Neurological deficits were measured by assessing mNSS score at day 1 after eMCAO. H and I, Sensorimotor function was measured using adhesive removal test and corner test until 28 d after eMCAO. Data are shown as mean±SEM; n=10 in saline group, 11 in tPA group, 10 in RP101075 group, 12 in tPA+RP101075 group, 10 in Bindarit group, and 10 in tPA+Bindarit group. Kruskal-Wallis test in E and F, 1-way ANOVA in G, and 2-way ANOVA in H and I. *P<0.05, **P<0.01. mNSS indicates modified neurological severity score. exogenously administered tPA into the perivascular space. 10 However, according to a recent meta-analysis of individual patient data from 6756 patients, fatal intracranial hemorrhage risk was similar irrespective of treatment delay and stroke severity, suggesting that intravascular effects of tPA may, at least in part, play a role in HT formation. 31 This study presents the first definitive evidence that tPA thrombolysis swiftly mobilizes neutrophils and T cells to accumulate in the cerebrovascular compartment. These cells then act on endothelial cells and induce BBB disruption. The combination of augmented focal inflammation after ischemia together with the direct damage exerted by tPA-mobilized immune cells results in grave consequences for the brain vasculature, leading to hemorrhagic complications.
Understanding the inflammatory mechanisms governing the emergence of HT in ischemic stroke provides an opportunity to counter this devastating complication of tPA. In the present study, we have adopted 2 approaches to decouple the interaction between tPA and immune cells via interference of cell migration and targeting a receptor that conjugates tPA. The identification of annexin A2 as a mediator between tPA and immune cells suggests that targeting annexin A2 may block the action of tPA on immune cells. In addition to mediating adverse effects on immune cells, annexin A2 also plays an important role in accelerating the thrombolytic effects of tPA by bridging tPA and fibrin. We previously found that recombinant annexin A2 enhances tPA-mediated thrombolysis. 4,32 In addition to facilitating thrombolysis, our new finding suggests that recombinant annexin A2 could simultaneously abrogate the binding of tPA to annexin A2 expressed on immune cells, thus suppressing leukocyte mobilization post-thrombolysis. In line with this postulate, our previous studies have demonstrated that recombinant annexin A2 reduces HT, preserves BBB integrity, and attenuates local inflammation. 32,33 The fact that inhibiting the transmigration of lymphocytes and neutrophils reduces tPA-related brain hemorrhage is of clinical impact. Emerging evidence indicates that interactions between lymphocytes and endothelial cells foster microvascular dysfunction and secondary infarct growth after brain ischemia. 34 Activated CD4 + and CD8 + T cells are sources of IFN-γ (interferon-γ), perforin, IL (interleukin)-23, IL-17, and other inflammatory factors that lead to neuronal cell death and BBB disruption. 35,36 Depletion of lymphocytes or inhibition of their egress from lymphoid organs attenuates ischemic injury. 26,37 Here, we demonstrate that tPA administration accelerates the recruitment of T cells to the brain vasculature, contributing to subsequent BBB disruption and hemorrhage, and inhibition of lymphocyte egress mitigates tPA-associated brain hemorrhage. It is also noteworthy that neutrophils are among the first peripheral cell populations to respond to brain ischemia, and they exacerbate ischemic brain injury via release of proteases such as MMPs and formation of extracellular traps. 17,38 One recent study suggests that neutrophil adhesion to brain endothelium leads to stalled blood flow in cerebral microcirculation, 39 highlighting a detrimental role of neutrophils in microvascular dysfunction. In line with prior reports, the present study demonstrates that tPA promotes neutrophil migration via a CCL2-CCR2 pathway, and inhibition of CCL2 synthesis reduced neutrophil transmigration following tPA treatment, thus attenuating brain hemorrhage.
Recently completed phase II clinical trials have evaluated the safety and efficacy of combining the immune modulator fingolimod with tPA thrombolysis in the treatment of patients with acute ischemic stroke. 22,29 The outcome of these trials indicates that the combination is safe and potentially reduces HT and improves outcome, even when administered beyond the approved 4.5-hour time window for tPA. 22,29 The present study provides novel insight into the underlying mechanism of the improved efficacy observed in these trials and rationalizes large-scale, controlled clinical trials to confirm the benefits of adjuvant immune therapy in future clinical trials of thrombolysis.
In all, the present study establishes that tPA thrombolysis induces a rapid surge of neutrophils and T cells in the circulation within an hour of administration, which is an active contributor to postthrombolysis HT but not a secondary response to tPA-associated adverse events. The direct effects of tPA on immune cell are mediated by annexin A2 that is the molecular switch to turn on the rapid response of neutrophils and T cells to tPA administration. Our study suggests that precise modulation of peripheral immune components could be an attractive pathway to prevent tPA-associated HT.
Several questions remain. First, it is uncertain whether inhibiting the transmigration of neutrophils or T cells is sufficient to curb HT in a clinical setting. Second, the ideal patient population to benefit from immune modulation as an adjunct therapy to tPA, as well as the optimal timing and duration of therapy, remains unclear. Only male animals were used in the present study. Although we found a similar responsiveness of circulating immune cells to tPA treatment in male versus female patients with ischemic stroke, the sex difference in tPA-related immune activation requires future investigations. Third, in addition to tPA, an altered peripheral environment after brain ischemia and immune-endothelial cellular interactions may also contribute immune cell action in conjunction with tPA. Additionally, the question of whether inhibition of neutrophils and T cells would increase the risk of infection remains unanswered, as is whether we can disassociate the ability of annexin A2 to mediate the thrombolytic effect of tPA and its action on immune cells to prevent HT at a new level. Answers to these questions will likely pave the way to a novel pharmacological