tPA Mobilizes Immune Cells that Exacerbate Hemorrhagic Transformation in Stroke

Kaibin Shi1,2, Ming Zou1, Dong-Mei Jia1, Samuel Shi3, Xiaoxia Yang1, Qiang Liu1, Jing-fei Dong4, Kevin N Sheth5, Xiaoying Wang6, Fu-Dong Shi1,2*

1Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin 300052, China;2China National Clinical Research Center for Neurological Diseases, Jing-Jin Center for Neuroinflammation, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China;3Neuroscience graduate program, Arizona State University, Tempe, AZ 85281, USA;4BloodWorks Northwest Research Institute, Division of Hematology, Department of Medicine, School of Medicine, University of Washington, Seattle 98102, WA, USA;5Department of Neurology, Yale University School of Medicine, New Haven 06510, CT, USA;6Department of Neurosurgery, Clinical Neuroscience Research Center, Tulane University School of Medicine, New Orleans 70112, LA, USA.

Running title: tPA-Induced Immune Invasion in Brain Hemorrhage

Subject Terms:
Inflammation Ischemic Stroke Translational Studies

Address correspondence to:
Dr. Fu-Dong Shi Department of Neurology
Tianjin Neurological Institute
Tianjin Medical University General Hospital Tianjin 300052, China
[email protected].

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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.

ImageMethods 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.

Key words:
Tissue plasminogen activator, ischemic stroke, inflammation, hemorrhagic transformation, immune therapy, stroke, thrombolysis, immune system.

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Nonstandard abbreviations and acronyms
tPA tissue plasminogen activator
HT hemorrhagic transformation
BBB blood brain barrier
LRP low density lipoprotein receptor-related protein
NMDAR N-methyl-D-aspartate receptor
eMCAO embolic middle cerebral artery occlusion
NK cell natural killer cell
PBS phosphate-buffered saline
CCL2 C-C motif chemokine ligand 2
MMP matrix metallopeptidase
MRI magnetic resonance imaging
HGD hypoxia and glucose deprivation
MAPK mitogen-activated protein kinase
MIRB Molday ion rhodamine B
CCR2 C-C motif chemokine receptor 2


Tissue plasminogen activator (tPA) and endovascular thrombectomy are the two pillars of treatment for patients with acute ischemic stroke. However, only patients with large vessel occlusion have been shown to benefit from thrombectomy, which requires expertise and infrastructure that are not evenly distributed across centers1, 2. Within 4.5 hours of ictus, tPA remains the gold standard treatment for eligible ischemic stroke patients. However, due to the narrow therapeutic window and potential severe adverse events such as hemorrhagic transformation (HT) and malignant brain edema associated with the non-thrombolytic effect of tPA, fewer than 5% ischemic stroke patients benefit from tPA treatment2.

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 non-thrombolytic effects of tPA may contribute to this devastating complication. tPA binds several receptors, including annexin A2, low density lipoprotein receptor-related protein 1 (LRP1), and the N-methyl-D-aspartate receptor (NMDAR), resulting in differential downstream biological effects3-7. For instance, tPA compromises BBB integrity via LRP1 expressed on endothelial cells, microglia, and astrocytic endfeet5, 8, 9. tPA also activates platelet- derived growth factor-CC (PDGF-CC)10 and kallikrein11 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 lipopolysaccharide6 and also increases leukocytes infiltration into lung, kidney, and cremaster muscle during ischemia/reperfusion12-15. Among leukocyte subsets, neutrophils have been linked to intracerebral hemorrhage after thrombolysis in animal models16, 17 and patients with ischemic stroke18, 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 or consequence of tPA-related HT. Second, the direct effects of tPA on leukocytes and their contribution to HT after tPA thrombolysis are still unclear20. Third, in addition to neutrophils, the role of other leukocyte subsets such as lymphocytes remains elusive in tPA-related HT. To address these questions, we have carried out studies on ischemic

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stroke patients receiving tPA thrombolysis and a rat embolic middle cerebral artery occlusion (eMCAO) model.


A detailed methods section is provided in supplemental materials. Please refer to the Major Resources Table in the Supplemental Materials.

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


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 approximately 15% and 19%, respectively, as early as 1 hour after tPA administration compared to cell counts prior to tPA treatment and control patients (Supplemental Figure I). Further analysis of cellular subsets via flow cytometry revealed that neutrophils increased by 31%; T cells, including CD4+ 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 (NK) cells remained relatively stable before and after tPA treatment, these cell counts were also comparable to those of control patients (Supplemental Figure II).

tPA swiftly mobilizes neutrophils and T cells in ischemic stroke patients and in a rat model of focal embolic stroke.

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 (Supplemental Figure III). This model more accurately recapitulates the key components of thromboembolic stroke and subsequent tPA-induced thrombolysis in human ischemic stroke, relative to the intraluminal filament middle cerebral artery occlusion (MCAO) model21. In eMCAO rats, the count of circulating neutrophils was increased at 1 hour after tPA administration by approximately four-fold as compared to controls receiving saline. The increase of circulating neutrophils was sustained up to 12 hours post-thrombolysis (Figure 2A-B). CD4+ and CD8+ T cells were also increased by approximately two-fold in peripheral blood samples of eMCAO rats treated with tPA, with elevation of CD8+ T cells detected as early as 15 min after tPA administration (Figure 2B). A transient increase in B cell count was also observed following tPA treatment (Supplemental Figure IVB), this was not apparent in blood samples of stroke patients. On the other hand, monocytes and NK cells were not significantly impacted by tPA administration (Supplemental Figure IVA, C). In addition, no significant sex impact on the peripheral immune response of eMCAO rats after tPA thrombolysis was recorded (Supplemental Figure V).

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

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altered genes (upregulated: 1449 genes, downregulated: 1126 genes) after tPA thrombolysis as compared to eMCAO group receiving PBS. Moreover, 820 altered genes were identified in eMCAO+PBS group versus eMCAO+tPA group (Supplemental Figure VIA-B). Among the altered genes, we found a significant increase of genes related to neutrophil chemotaxis, activation and infiltration, including CCR2 and MMP9 (Figure 2C-D). 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 were 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 hemorrhagic transformation following tPA thrombolysis.

ImageWe next examined the destination of migrating immune cells following tPA thrombolysis in eMCAO rats. Compared to 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 brain-infiltrating neutrophils and T cells were associated with their corresponding counts in blood after tPA thrombolysis (Supplemental Figure VII). In addition, upregulation of CCR2, CXCR2, MMP9 and TLR4 was observed in brain-infiltrating neutrophils (Supplemental Figure VIII). 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-C).

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 post-thrombolysis 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 7-Tesla (7T) rodent magnetic resonance imaging (MRI) scanning using a T2* sequence, which is sensitive to brain hemorrhage22. 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 5-10 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 1-100 µg/mL tPA, flow cytometry analysis was performed to evaluate the expression of adhesion molecules in these cells. A time- and 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-C). 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 100µg/mL (Figure 4A), while the CD49d expression of T cells was not upregulated until 12 hours after tPA treatment (Figure 4B-C). 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 cells23. 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 in newly released Biotin- neutrophils versus old Biotin+ neutrophils (Supplemental Figure IX). Similarly, we observed upregulation of CD69 and CD49d in newly released Biotin- T cells versus old Biotin+ T cells

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(Supplemental Figure IX). 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 co-culture 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 (Supplemental Figure X).

Next, we sought to determine whether tPA-activated neutrophils or T cells exacerbate endothelial injury, the key pathologic mechanism underlying HT24. 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 glucose deprivation (HGD) for 4 hours, followed by co-culture with tPA-treated neutrophils or T cells. FITC-dextran that leaked from the upper chamber to the lower chamber was quantified to assess the permeability. Relative to baseline, HGD increased the diffusion of FITC-dextran as early as 1 hour after hypoxia induction. tPA-treated neutrophils or T cells exacerbated HGD-induced FITC-dextran diffusion by approximately two-fold, whereas untreated neutrophils or T cells produced only a trend towards 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-F). 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 (Supplemental Figure XI). We then examined the potential downstream pathways that mediate the effects of tPA on immune cells. KEGG pathway enrichment analysis revealed that MAPK 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-B), suggesting that MAPK pathway might mediate the activation of immune cells after tPA administration. Western blot analysis demonstrate 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 (Supplemental Figure XII). After labelling with Molday ION Rhodamine B (MIRB), 5 x 106 cells were injected via tail vein to recipient rats prior to eMCAO surgery and tPA administration (Figure 6A). MIRB is a fluorescence-conjugated nanoparticle that can be used to trace extrinsically transferred cells in vivo as we previously reported25. Compared cells transfected with control siRNA, annexin A2 knock down inhibited tPA-induced activation of p38 MAPK (Supplemental Figure XIII). The expression of CCR2 and CD49d on neutrophils and T cells was also reduced after annexin A2 knock down at 12 hours post-tPA administration (Figure 6D-E, G-J), together with reduced transmigration of these cells into the ischemic brain (Figure 6F, K). These findings suggest that the tPA receptor annexin A2 is required for the effects of tPA on peripheral immune cells.

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Suppression of the transmigration of neutrophils and T cells reduced tPA-associated hemorrhagic transformation 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 (Supplemental Figure XIV). We then tested two immune modulating drugs that have translational potential. RP101075 is a second generation of sphingosine-1-phosphate receptor (S1PR) modulator which selectively inhibits S1PR1-dependent lymphocytes egress from secondary lymphoid organs26. Compared to eMCAO rats receiving saline, eMCAO rats receiving tPA at 3 hours after ischemia had significantly increased brain hemorrhage at 24 hours post stroke (saline vs tPA: 5.6 ± 0.7 vs 14.7 ± 1.4 mm3) without amelioration of infarction (Figure 7A-F). In eMCAO rats receiving RP101075, we found significantly reduced numbers of circulating and brain-infiltrating T cells (Supplemental Figure XVA-B). RP101075 significantly reduced brain hemorrhage in eMCAO rats receiving tPA with a 46% reduction as compared to tPA-treated rats (tPA + RP101075 vs tPA: 7.9 ± 1.5 vs 14.7 ± 1.4 mm3). In addition, the infarct volume of tPA + RP101075 treated eMCAO rats was 40% smaller than that of tPA-treated rats (tPA + RP101075 vs tPA: 138.9 ± 12.7 vs 231.4
Image± 14.7 mm3).
Although tPA thrombolysis at 3 hours after eMCAO did not significantly improve the neurological deficit as compared to 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-I, Supplemental Figure XVI).

In addition, we examined whether inhibition of neutrophil migration using bindarit reduces tPA- associated hemorrhage. Bindarit is a CCL2 inhibitor that blocks the migration of myeloid cells including neutrophils27. 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 (Supplemental Figure XVC-D). Combination of bindarit with tPA reduced volume of brain hemorrhage by 34% compared to tPA treated rats at 24 hours post-ischemia (tPA + bindarit vs tPA: 9.7 ± 1.5 vs 14.7 ± 1.4 mm3). Infarct volume was reduced by 36% in rats receiving combined treatment of tPA and bindarit relative to rats receiving tPA alone (tPA + bindarit vs tPA: 146.2 ± 19.0 vs 231.4 ± 14.7 mm3). Bindarit also reduced acute neurological deficits at 24 hours after ischemia and improved sensorimotor function at 4 weeks (Figure 7G-I). 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.


Previous studies suggest that tPA upregulates the expression of MMPs on brain vascular endothelium8 and accelerates the degradation of the extracellular matrix of blood vessels, thereby contributing to HT following thrombolytic therapy. In addition to directly activating 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 (Supplemental Figure XVII).

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 approximately 5 minutes.

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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 hours2. 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, twenty-four hours has commonly been used as the time-point to assess the effects of tPA and related adverse events18, 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 prior to 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 stroke24, 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. Post-thrombolysis hemorrhage is the most feared complication of intravenous thrombolysis, with almost 50% mortality in cases with symptomatic hemorrhage following tPA24. HT occurs when the integrity of the endothelial cells lining brain vasculature and BBB becomes compromised24. Endogenous tPA engendered during brain ischemia also increases the permeability of BBB, which may prime microvessels for the deleterious extra-vascular effects of therapeutically-administered exogenous tPA5, 8, 10. Increased BBB permeability prior to tPA treatment, which is determined by stroke severity and time to treatment, is thought to promote leakage of exogenously- administered tPA into the perivascular space10. However, according to a recent meta-analysis of individual patient data from 6,756 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 formation31. 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 two 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 thrombolysis4, 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 inflammation32, 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 ischemia34. Activated CD4+ and CD8+ T cells are sources of IFN-γ, perforin, IL-23, IL-17, and other inflammatory factors which lead to neuronal cell death and BBB disruption35, 36. Depletion of lymphocytes or inhibition of their egress from lymphoid organs attenuates ischemic injury26, 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 very first peripheral cell populations to

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respond to brain ischemia, and they exacerbate ischemic brain injury via release of proteases such as MMPs and formation of extracellular traps17, 38. One recent study suggests that neutrophil adhesion to brain endothelium leads to stalled blood flow in cerebral microcirculation39, 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 acute ischemic stroke patients22,
29. The outcome of these trials indicate that the combination is safe and potentially reduces HT and improves outcome, even when administered beyond the approved 4.5-hour time window for tPA22, 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.

ImageIn 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 post-thrombolysis 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 hemorrhagic transformation 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 as well as 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 in order to prevent HT at a new level. Answers to these questions will likely pave the way to a novel pharmacologic strategy which attenuates HT, a major limitation of tPA-mediated thrombolysis in stroke.

F.-D. S. and X. W. formulated the concept; D.-M. J., X. Y., and M. Z. enrolled stroke patients and acquired the clinical data; K. S., X. Y. and S. S. conducted animal and in vitro experiments; K. S., X. Y., D.-M. J., and F.-D. S. analyzed the results. F.-D. S., K. S., Q. L., X. W., K.N. S. and J.-f. D. interpreted the results.
K. S., Q. L., S. S., J.-f. D. and F.-D. S. drafted the manuscript.

We thank our patients for their participation in this study. We thank S. Gao, L. Zhang, D.-C. Tian for patient recruitment, and H. Li, Y. Kong for technical assistance. This work was supported in part by National Science Foundation of China (91642205, 81830038 and 81701176); the Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, China; and National Key Research and Development Program of China (2018YFC1312200).

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