Role of tissue plasminogen activator in clinical aggravation of experimental autoimmune encephalomyelitis and its therapeutic potential

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Role of tissue plasminogen activator in clinical aggravation of experimental autoimmune encephalomyelitis and its therapeutic potential Tehila Mizrachia, Devorah Gur-Wahnona, Abd Al-Roof Higazib, Talma Brennera,

⁎

a Laboratory of Neuroimmunology, Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah-Hebrew University Medical Center, PO Box 12000, Jerusalem, Israel b Department of Biochemistry, Hadassah-Hebrew University Medical Center, PO Box 12000, Jerusalem, Israel

A R T I C LE I N FO

A B S T R A C T

Keywords: Tissue plasminogen activator (tPA) Multiple sclerosis T-regulatory cells Central nervous system inflammation Tissue plasminogen activator variants

Tissue plasminogen activator (tPA), a component of the plasminogen activator (PA) system, is elevated in inflammatory neurological disorders. In the present study, we explored the immunomodulatory activity of tPA in experimental autoimmune encephalomyelitis (EAE). The EAE was treated with two catalytic inactive tPA variant proteins: S(481)A and S(481)A + KHRR (296–299)AAAA. EAE-induced tPA−/− mice presented with markedly more severe disease than wt EAE mice. Further, treatment with tPA variants, demonstrated a significant suppression of disease severity in tPA−/− and wt mice. Immunological evaluation showed that specific T-cell reactivity was markedly reduced in the tPA−/− animals, as indicated by decreased T-cell reactivity and reduction in T-regulatory cells. The current findings indicate that tPA plays a role in the pathogenesis of EAE. Moreover, successful amelioration of EAE was achieved by administration of tPA variant proteins. This might mean that these proteins have potential for the immunomodulation of neuroinflammation.

1. Introduction Multiple sclerosis (MS) is a prototypical inflammatory demyelinating disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for MS studies. EAE is a complex condition that mimics the key pathological features of MS through the interaction between a variety of immunopathological and neuropathological mechanisms: inflammation, demyelination, axonal loss and gliosis [1]. This model has been instrumental to the development of new therapies for MS [1], and this is the model that will be employed in the present study. Extracellular proteolysis is critical for physiological and pathophysiological processes such as inflammation, tumor invasion, tissue repair and excitoxicity [2,3]. The plasminogen activation (PA) system is one of the major actors in extracellular proteolysis [4–6]. Tissue plasminogen activator (tPA) is an important component of the PA system that performs the cleavage of the zymogem plasminogen to its active form [7]. tPA participates in the activation of various zymogens and

growth factors and is involved in the homeostasis of blood coagulation [6]. Plasminogen receptors are expressed on various cell types such as monocytes, granulocytes and endothelial cells [8]. Accumulating evidence suggests that the PA system plays an important role in the pathogenesis of MS and EAE [9–11]. Specifically, PA activity is considered important in the disturbance of the blood – brain barrier (BBB) and subsequent leukocyte migration, which leads to inflammation and myelin breakdown [10]. Further, there is evidence that tissue plasminogen activator (tPA), is involved in the regulation of inflammatory autoimmune diseases [12–18]. For instance, tPA promoted infiltration of macrophages and other leukocytes in models of acute and chronic kidney injury as well as activated macrophages in acute brain injury [19]. Accordingly, East et al. [10] reported more severe EAE in tPA−/− mice than in wt EAE mice, as indicated by incomplete recovery and increased neurological deficit. As additional evidence, our previous study showed that mice lacking tPA develop more severe experimental autoimmune myasthenia gravis (EAMG) [20]. In agreement with these findings, in MS patients, too, a significant increase in the level of

Abbreviations: APC, antigen-presenting cell; BBB, Blood-brain barrier; CFA, Complete Freund's adjuvant; CNS, central nervous system; EAE, Experimental autoimmune encephalomyelitis; FCS, fetal calf serum; INF, Interferon; IL, Interleukin; ko, Knockout; LNC, lymph-node cell; MOG, Myelin oligodendrocyte glycoprotein; MS, Multiple sclerosis; PA, Plasminogen activator; tPA, tissue plasminogen activator, PAI-1, Plasminogen activator inhibitor-1; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor; wt, wild type ⁎ Corresponding author. E-mail address: [email protected] (T. Brenner). https://doi.org/10.1016/j.cellimm.2020.104040 Received 8 December 2019; Received in revised form 5 January 2020; Accepted 9 January 2020 0008-8749/ © 2020 Published by Elsevier Inc.

Please cite this article as: Tehila Mizrachi, et al., Cellular Immunology, https://doi.org/10.1016/j.cellimm.2020.104040

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2.3. tPA variants and PAI-1 treatment

endogenous tPA antigen was found [7,21–23], along with high levels of plasminogen activator inhibitor type-1 (PAI-I) in the cerebrospinal fluid [22]. We have previously demonstrated the involvement of urokinase plasminogen activator (uPA) and its receptor (uPAR) in neuroinflammation. We reported that deficiency of these components (uPA and uPAR) in mice with EAE led to the aggravation of the disease in the form of increase in inflammation and axonal damage, increase in histopathological manifestations, and increased activation of microglia/ macrophages in the CNS, while treatment with a PAI-1-derived peptide ameliorated EAE [11]. We have extended our observations to another autoimmune model EAMG, and found that mice lacking tPA developed a more severe EAMG and treatment with PAI-1-derived peptides led to suppression of the disease [20]. These findings in animals lacking tPA formed the basis of the present study, which aimed to examine the potential of treating EAE with tPA. Recombinant tPA is widely used as a thrombolytic agent in acute myocardial infarction and in ischemic stroke; however, it can be administered only once as its catalytic activity causes bleeding [11,24] In order to overcome this limitation, in the present study, we used two treatment approaches to increase tPA level/activity: 1) The tPA variants S(481)A, containing one mutation in the catalytic site, and S (481)A + KHRR(296–299)AAAA, containing an additional mutation at the plasminogen inhibitor docking site. These proteins have anti-inflammatory effects but no catalytic activity and can be injected several times without causing severe hemorrhage. [25] 2) treatment with an 18-amino acid peptide derived from PAI-I that binds to tPA and inhibits the binding of the endogenous PAI to tPA, thus increasing the activity of tPA [25]. In the present study, we induced EAE in wild-type (wt) and tPA−/− (tPA knockout) mice. The tPA−/− mice developed a more severe form of EAE that was accompanied by a reduction in T-cell reactivity and the number of T regulatory cells. Further, treatment with tPA variant proteins suppressed disease severity both in wt and tPA−/− mice. Altogether, the findings indicate that exogenous intervention to correct tPA dysfunction might be beneficial in the immunomodulation of neuroinflammation.

tPA-S(481)A substitution of serine at position 481 with alanine resulted in the formation of a mutant tPA variant without catalytic activity. Further, a double-mutant tPA variant with the same serine substitution mutation and the substitution of KHRR with AAAA position 296–299 (PAI-I docking site) was also created [25–27]. The tPA-S (481)A variant, the S(481)A + KHRR(296–299)AAAA variant, or the placebo were injected intraperitoneally twice daily at a dose of 0.5 mg/ kg [28], from 1 day prior to EAE induction, for 7 consecutive days. The mice were then observed and scored as described for the wt experiment. The EAE mice were also treated with an 18aa peptide, Ac-RMAPEEIIMDRPFLYVVR-amide, derived from the endogenous PAI1 protein (PAI-1-dp) [11; 24]. PAI-1-dp was injected intraperitoneally twice daily at dose of 0.5 mg/kg, starting from 1 day before EAE induction, for 7 consecutive days. The control mice were administered an equal dose of a placebo. Mice from both groups were observed and scored as described in the wt experiment. 2.4. Lymphocyte proliferation assay “Pooled lymph node cells (LNCs) were prepared from the inguinal, axillary, and mesenteric lymph nodes or spleens derived from naive mice or from mice that had been subcutaneously inoculated 9 days earlier with the MOG35–55 peptide in CFA. The in vitro lymphocyte response was assayed in triplicate wells of 96-well flat-bottom microtiter plates. A total of 2 × 105 LNCs, suspended in 0.2 ml RPMI supplemented with 5% fetal calf serum (FCS), was added to each well with or without 100 μg/ml of the MOG35–55 peptide. At 48 h after seeding, 1 μCi [H3]thymidine (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) was added to each well, and the plates were incubated for an additional 18 h. The plates were then harvested with a semi-automated harvester onto a glass fiber filter, and radioactivity was assessed with a liquid scintillation assay” [11]. 2.5. Cytokine secretion assay

2. Materials and methods

The culture media containing splenocytes or LNCs were incubated in the presence of MOG35-55, anti-CD3 antibody or lipopolysaccharide (LPS). Supernatant was collected at different time points: at 24 h for the IFNγ and IL-17 assay, and at 72 h for the IL-10 assay. The cytokine concentrations were determined with specific ELISA kits (Biolegend, San Diego, CA).

2.1. Mice Female (7- to 8-week-old) C57BL/6 mice were purchased from Harlan Laboratories, Rehovot, Israel. The animals were housed under specific pathogen-free (SPF) conditions at the animal facility of the Hebrew University Medical School, Jerusalem, Israel, in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. tPA−/− mice against a C57BL/6 background were purchased (Jackson Laboratories), bred at the animal facility of the Hebrew University Medical School, and housed under SPF conditions.

2.6. Flow cytometry analysis For leukocyte surface marker determination, pooled LNCs or splenocytes were obtained from wt and tPA−/− mice (as described in the mouse lymphocyte proliferation assay). Cell suspensions were prepared as described previously [29]. For immune phenotyping, the following antibodies were used: anti-CD4 FITC, anti-CD8 FITC, anti-CD3 FITC, anti-CCR5 PE, anti-CD19 FITC, and anti-CD11b PE (all from eBioscience, San Diego. CA). Stained cells were counted with a FACS Calibur flow cytometry kit (FC500, Beckman Coulter). Regulatory T cells were stained using a regulatory T-cell staining kit (w/PE FoxP3 FJK-16 s, FITC CD4, APC CD25) (eBioscience, San Diego, CA). FOXP3 FJK-16 antibodies were used for intracellular staining for FOXP3 expression. Stained cells were counted with the FACS Calibur kit (FC500, Beckman Coulter) [20].

2.2. EAE induction and clinical evaluation EAE was induced in 8-week-old female C57BL/6 mice by subcutaneous administration of 250 μg MOG35–55 peptide (synthesized by Sigma Laboratories, Israel) into the left paralumbar region. The MOG35–55 peptide had been emulsified in complete Freund’s adjuvant (CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis. Immediately after this injection and at 48 h after the injection, the mice were intraperitoneally administered 200 ng of the pertussis toxin (List Biological Industries, San Diego CA, USA). All the animals were examined daily and evaluated for clinical signs of disease. The clinical status of the mice was graded as follows: 0, without clinical disease; 1, tail weakness; 2, hind limb weakness sufficient to impair righting; 3, single-limb plegia; 4, paraplegia with forelimb weakness; 5, quadriplegia; 6, death. According to the ethical requirements, mice that reached stage 4 were euthanized [11].

2.7. Antigen presentation assay Antigen-presenting cells (APCs) were derived from the spleens of wt and tPA−/− naive mice. Before co-culture with responder T cells, the splenocytes were irradiated at 3000 rad, so as to ensure that the proliferation in the co-culture assays could be attributed solely to the 2

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responder T cells. The responder cells were obtained from MOG35–55stimulated LNCs derived from MOG35–55-immunized wt mice. Pooled LNCs were prepared from inguinal and axillary lymph nodes from mice, 9 day post EAE immunization. Cells were plated in a concentration of 2 × 106 LNCs/ml in a 24 well plate, with 50 µg/ml MOG 35-55 for 4 days, after which the medium was replenished and supplemented with IL-2 (20 μg/ml) for an additional 5 days. For the antigen presentation assay, 50,000 responder T cells (per well) were co-cultured with 300,000 irradiated splenocytes derived from wt or tPA−/− naive mice in the presence of MOG35-55 (50 μg/ml) for 72 h. The cells were cultured in RPMI medium containing 10% FCS supplemented with 5 × 10−5 mol/l of 2-ME, 1 mmol/l sodium pyruvate, 1/100 nonessential amino acids, 2 mmol/L L-glutamine 2, and 100 U of penicillin/ ml. All the media and reagents were purchased from Biological Industries, Beit Haemek, Israel. Responder T-cell proliferation was assessed by [H3] thymidine incorporation [11]. 2.8. Preparation of RNA and cDNA “Total RNA was prepared using a 5 Prime RNA kit (GmBh Hamburg, Germany). cDNA was prepared from 1 µg of total RNA, using a High Capacity Reverse Transcription kit (Applied Biosystems Grand Island NY, USA)” [20]. 2.9. Quantitative real-time PCR A total volume of 20 μl of reaction mixture containing 50 ng cDNA; 300 nM of the appropriate forward and reverse primers (Sigma Genosys Ltd., Cambridgeshire, UK); and 10 µl of the Master Mix buffer containing nucleotides, Taq polymerase, and SYBR green (SYBR Green Master Mix, Applied Biosystems) was prepared. Gene amplification was carried out using the GeneAmp 7000 Sequence Detection System (Applied Biosystems). The amplification protocol was 10 min at 95 °C followed by 40 cycles of a two-step loop of 20 s at 95 °C and 1 min at 60 °C. The results are expressed as relative quantification (RQ) the fold increase of gene expression in MOG35-55-treated cells relative to the gene expression in control cells. Gene expression was normalized to the expression of the HPRT gene, which was not altered under the experimental conditions [20]. The sequences of the primers used were as follows. HPRT Forward: 5′-TCCTCCTCAGACCGCTTTT-3′ Reverse: 5′-CCTGGTTCATCATCGCTAATC-3′ GATA-3 Forward: 5′-GCAGAAAGCAAAATGTTTGCTTC-3′ Reverse: 5′-GAGTCTGAATGGCTTATTCACAAATG-3′ RORγt Forward: 5′-CCCTGGTTCTCATCAATGC-3′ Reverse: 5′-TCCAAATTGTATTGCAGATGTTC-3′ T-bet Forward: 5′-CAGTTCATTGCAGTGACTGCCTAC-3′ Reverse: 5′-CAAAGTTCTCCCGGAATCCTTT-3′ FoxP3 Forward: 5′-AGAAGCTGGGAGCTAT-3′ Reverse: 5′-GCTACGCTGCAGCAAG-3′

Fig. 1. Role of tPA in EAE and effect of treatment with tPA variant proteins. (A) Clinical severity of EAE in tPA−/− mice and control wt mice. The mean clinical severity differed significantly between the two groups (p < 0.05). The results are expressed as the daily mean clinical score of each group (wt, n = 22; tPA−/ − , n = 9). (B) EAE-induced wt mice were treated with the tPA variants S(481)A or S(481)A KHRR(296–299)AAAA or PAI-1 dp or placebo from 1 day prior to induction of EAE for seven consecutive days. tPA variant proteins were administered intraperitoneally at 0.5 mg/kg twice a day, and the EAE clinical score was markedly reduced (placebo, n = 10; S(481)A, n = 9; S(481)A KHRR (296–299)AAAA, n = 10; PAI-I d.p, n = 10) The clinical severity differed significantly between the two variant protein groups and the placebo group (p < 0.05). The results are expressed as the daily mean clinical score for each group and represent the mean values of three different experimental groups. (C) EAE-induced tPA−/− mice treated with the tPA variants or the placebo, as described for the wt mice (n = 6 in each group). The clinical severity differed significantly between the two groups (**p < 0.001), and the scores shown are the mean values of the two groups.

2.11. Statistical analysis The data were analyzed using Student’s t-test and one-way ANOVA, according to Dunnett, and the Fisher Exact Test. p < 0.05 was considered significant. 3. Results 3.1. Role of tPA in EAE and effect of treatment with tPA variant proteins

2.10. Histopathology analysis To assess the involvement of tPA in EAE, we induced EAE in tPA−/− mice and wt mice and compared the clinical severity. As shown in Fig. 1A and Table 1, in the absence of tPA, the clinical score was significantly higher: the tPA−/− mice showed a mean disease score of 4.2 ± 0.3 and the wt mice had a score of 1.7 ± 0.2 (a 2.4-fold difference). In addition, the cumulative score was 76.8 ± 9.4 in the tPA−/− mice and 29.4 ± 0.4 in the wt mice (p < 0.0001) and the mean maximum score of the wt was 3.6 ± 0.4 and the mean maximum severity of the tPA−/− group was 5.1 ± 0.4 (p < 0.05). Treatment

On the day of sacrifice, lumbar spinal cords with predominant inflammatory foci were harvested, fixed in 4% paraformaldehyde, dehydrated and then embedded in paraffin. Longitudinal sections spanning the majority of the length of the spinal cord and containing both gray and white matter were stained with hematoxylin-eosin as previously described [30]. The slides were evaluated under a light microscope (Axioplan-2; Zeiss), and inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section [30]. 3

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mice) (Fig. 2A). After treatment with the single-mutant, double-mutant and PAI-1-dp, the neuropathological features improved and the number of inflammatory foci significantly decreased by 50%, 80% and 60% respectively (p < 0.05) (Fig. 2B). (Comparing the number of inflammatory foci of PAI-1-dp treated mice versus the tPA variants treated mice, did not show statistical significant p > 0.05). Thus, the extent of neuroinflammation can be suppressed by administration of tPA variants or by increasing tPA reactivity, through neutralization of the endogenous inhibitor PAI-1.

Table 1 Influence of tPA deficiency and treatment with tPA variant proteins or PAI-I-dp on EAE. A

wt tPA−/−

mean severity

cumulative score

max score

1.7 ± 0.2 4.1 ± 0.3**

25.8 ± 3.5 76.8 ± 6.9*

3.6 ± 0.4 5.1 ± 0.4*

B

3.3. Role of tPA in functional T-cell reactivity and antigen presentation Placebo S(481)A S(481)A, KHRR (296–299)AAAA PAI-I

mean severity

Cumulative score

max score

2.1 ± 0.3 1.1 ± 0.3* 0.7 ± 0.2*

47.2 ± 7.4 24.3 ± 7.0* 17.1 ± 5.9*

2.7 ± 0.3 1.6 ± 0.4 1.3 ± 0.4*

0.35 ± 0.05**

10.9 ± 1.2*

1.4 ± 0.1*

To further understand the mechanism by which tPA influences immune reactions, we examined T-cell responses, that is, proliferation, cytokine secretion and antigen presentation. Intriguingly, in contrast to disease severity, the reactivity of T cells derived from tPA−/− mice and stimulated ex vivo with the encephalitogenic MOG35-55 peptide, was reduced by 57% compared to the wt cells (Fig. 3A, p < 0.0001), with reduced secretion of pro-inflammatory cytokines. An 82% reduction in INFγ secretion, 92% reduction in IL-17 secretion, and 53% reduction in the secretion of the anti-inflammatory cytokine IL-10 was observed (Fig. 3B and C). The ability of APCs derived from tPA−/− mice to activate MOG35-55specific T cells was impaired. As shown in Fig. 4A, in the presence of APCs from tPA−/− mice, T-cell proliferation was significantly reduced by 45% as compared with APCs derived from wt mice. This indicates that APCs derived from tPA−/− mice have reduced antigen-presentation capacity. Another surprising outcome was the significant decrease in the number of CD11b macrophages and CCR5-positive cells (by 19% and 34% respectively) in the tPA−/− mice (Fig. 4B), without any significant change in the number of CD4-positive cells.

C

wt tPA−/− placebo tPA−/− S(481)A

mean severity

cumulative score

max score

1.7 ± 0.2 2.2 ± 0.2 1.3 ± 0.3*

48.6 ± 6.9 64.2 ± 10.4 35.2 ± 9.3

2.3 ± 0.2 3.4 ± 0.5 2.1 ± 0.5

Mice were intraperitoneally administered the tPA variant proteins, PAI-1-dp or saline (control group) at a dose of 0.5 mg/kg twice a day for 7 consecutive days. The scores indicate the average daily clinical score of each group. The cumulative score is calculated as the number of days that the animal was sick multiplied by the clinical score. The max score indicates the average maximum clinical score for each mouse in each group. In Table 1A. the number of wt mice, n = 22; tPA−/−, n = 9; In Table 1B. the number of mice treated with placebo, n = 10; treated with S(481)A, n = 9; the number of mice treated with S(481)A KHRR(296–299)AAAA, n = 10; the number of mice treated with PAI-I d.p, n = 10; In Table 1C. The number of wt mice, n = 22; the number of tPA−/− treated with placebo, n = 6; the number of tPA−/− treated with S(481)A, n = 6) (*p < 0.05, **p < 0.001).

3.4. T-cell Profile and T-regulatory cells Based on the paradoxical T-cell findings in the tPA−/− mice, we decided to examine the immunological profile of these mice in the naïve state as compared with naïve wt mice. No significant differences were found in the expression of CD4, CD8, CD3, CD25, and CD19 in the naïve state (Table 2). Therefore, we examined whether tolerance is impaired in the tPA−/− mice. To this end, we determined the number of T-regulatory cells that are pivotal in maintaining self-tolerance. We quantified the number of CD4 + CD25 + FOXP3 cells in tPA−/− and in wt mice (Fig. 5A). Naive tPA−/− mice had a significant reduction of 32% in Tregs compared with naïve wt mice. After induction of EAE, the reduction was greater at 42% and was correlated with disease severity (Fig. 5B). In addition, according to the results of RT-PCR, the mRNA expression of FOXP3 in the tPA−/− EAE mice was 63% lower than that in the wt EAE mice (Fig. 5C). There was no significant difference in the number of Treg cells after treatment with tPA variant proteins (data not shown). However, after treatment with PAI-1-dp, the number of T-regs was significantly increased by 25% (Fig. 5D).

with the tPA variant S(481)A or (S481)A + KHRR(296–299)AAAA was highly beneficial. These variant proteins lack the catalytic activity as a result of the substitution of the serine in position 481 with alanine and, thus, can be administered several times and for a longer period. The treatment protocol consisted of 14 injections administered intraperitoneal twice a day, starting from 1 day before EAE induction for 7 days, at a dose of 0.5 mg/kg (Fig. 1B and Table 1). Treatment with tPA- S(481)A and with the protein that contains the two mutations, S (481)A + KHRR(296–299)AAAA, remarkably reduced mean disease severity by 48% and 67% respectively (p < 0.01). In addition, treatment with PAI-1-dp, the 18-amino acid peptide derived from the PA inhibitor that was designed to bind the endogenous inhibitor, resulted in an increase in the activity of tPA and significant amelioration of the disease (Fig. 1B). Treatment with this peptide reduced the mean disease severity by 84%, being the most effective therapy in our experiments, related to the two variant tPA proteins. The difference between the mean disease scores of the PAI-1-dp treated mice versus the tPA-S (481)A or the S(481)A + KHRR(296–299)AAAA was statistical significant (p < 0.05). Further, we tested if treatment with tPA variant can reverse the ko mice phenotype. Interestingly, treatment of EAE tPA−/− mice with tPA-S(481)A reduced disease severity by 41%, Fig. 1C and Table 1 show that this variant can compensate the lack of tPA in the ko mice.

3.5. Immunological parameters following treatment with the tPA variant S (481)A in wt EAE-induced mice In agreement with the clinical observations, the mice that were treated with tPA-S(481)A or with PAI-I dp developed markedly and significantly less severe disease (Fig. 1B). Comparison of T-cell reactivity (of cells derived from the spleen) in the tPA-S(481)A-treated mice and in PAI-I dp treated mice, with that in placebo-treated mice revealed that there was a reduction in T-cell reactivity in response to the encephalitogenic MOG35-55 peptide. As shown in Fig. 6A and B, splenocyte proliferation was decreased by 34% following tPA-S(481)A treatment (Fig. 6A) and by 66% following PAI-I dp treatment (Fig. 6B).

3.2. Neuropathological assessment of neuroinflammation in EAE mice The disease severity observed in the mice was consistent with the neuropathological findings: in tPA−/− mice, which had greater disease severity, mononuclear cell infiltration as well as the number of foci were higher (4.9-fold increase in the number of foci in the tPA−/− 4

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Fig. 2. Neuropathological features of spinal cords in EAE mice treated with the tPA variants or PAI-I. (A) tPA−/− mice exhibited an almost five-fold increase in the number of neuroinflammatory foci (n = 3 for each group, *p < 0.05). (B) Treatment with tPA variant proteins or PAI-1 improved CNS inflammation and reduced the number of EAE lesions. (C) Placebo, (D) tPA−/−, (E), (F) S(481)A,KHRR(296–299)AAAA, (G) PAI-I dp. Representative images (hematoxylin and eosin staining) at 40× magnification. The spinal cords of the mice were removed at day 30 after EAE induction. The arrows indicate the foci. Fig. 3. Reduction in the T-cell reactivity of tPA−/− mice to MOG35–55. (A) Proliferation of lymphocytes from tPA−/− and wt mice was assessed based on [3H] thymidine incorporation in the presence of MOG35-55. (B) The interferon (IFN)-γ, IL-17 and (C) IL-10 levels were measured by ELISA, and the results are expressed as the mean ± SE value of three separate experiments (n = 9 for each group) (*p < 0.05).

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4. Discussion The findings of the present study show that tPA plays an important role in autoimmune responses in EAE. While mice lacking tPA present with more severe disease, administration of external tPA ameliorated the disease. The treatment of EAE mice with tPA variant proteins lacking catalytic activity ameliorated the disease in wt and even in the tPA−/− mice. Recombinant tPA is widely used as a thrombolytic agent in acute myocardial infarction and in ischemic stroke; however, it can be administered only once as its catalytic activity causes bleeding [11,24] In order to overcome this limitation, in the present study, we used two treatment approaches with tPA variants proteins and with an 18-amino acid peptide derived from PAI-I that binds to tPA and inhibits the binding of the endogenous PAI to tPA, thus increasing the activity of tPA. The tPA variants S(481)A, containing one mutation in the catalytic site, and S(481)A + KHRR(296–299)AAAA, containing an additional mutation at the plasminogen inhibitor docking site, were used, as these proteins have anti-inflammatory effects but no catalytic activity and can be injected several times without causing severe hemorrhage. Treatment with these variants ameliorated EAE and had an immunomodulatory effect. Similarly, the treatment with PAI-I dp was also highly effective (even more than the tPA variants). Our findings are in agreement with the study by East et al. which showed that mice lacking PAI-I developed milder EAE without clinical relapse and an overall reduction in neuroinflammation [31]. In contrast to our findings, Dahl et al. reported that neuronal overexpression of tPA (14-fold higher) did not endow any protection against EAE in mice [17]. This discrepancy is probably due to the expression level that is too high to be beneficial. There is evidence that in vitro low concentrations of tPA are protective [32,33] however, it seems that higher levels are toxic [34,35]. In the present study, the neuropathological findings for the mice were consistent with the clinical severity scores. tPA−/− mice showed an increase in the amount of inflammatory foci in the spinal cord, and treatment with the tPA variant proteins or PAI-I-dp resulted in a significant reduction in CNS neuroinflammation, within the same line of findings of our previous study with uPA and uPAR deficient mice [11]. Similarly, East et al also reported that during EAE tPA−/− and uPAR−/ − mice present with more severe neuropathological findings than the wt mice [10]. Several previous studies have evaluated the onset and severity of EAE in mice lacking tPA. While Lu et al 2002 reported that tPA−/− developed more severe form of EAE, with delayed disease onset [18], two later studies, East et al (2005) and Dahl et al (2016), reported that tPA−/− present with a more severe form of EAE, with earlier onset, similar to our results [10]. The reason for these opposing results regarding the timing of disease onset is unknown, although different mouse sources may have contributed to this [10; 17; 18]. Nevertheless, all those studies including ours conclude that tPA deficiency cause greater CNS damage and increased disease severity. Our present study focused on testing the effects of tPA variant proteins on the clinical outcome and the pathogenic events during EAE. Thus, treatment protocol was initiated one day prior to disease induction and lasted for 7 consecutive days and stopped before the appearance of clinical signs. Under these conditions, the disease was significantly ameliorated. However, we have not studied the effectiveness of these proteins on reversing ongoing disease. These experiments are plane to be performed in order to support the therapeutic potential of these compounds. A surprising finding in our study was that the tPA−/− EAE mice with severe disease exhibited a lower immune response, as indicated by the decrease in the number of proliferative lymphocytes as well as reduction in cytokine secretion. The finding is contradictory because severe disease is usually associated with high lymphocyte activity, but in our study showed the opposite results. However, it should be noted that the PA system is considered to be both pro-inflammatory and anti-

Fig. 4. Reduced antigen presentation in tPA−/− EAE mice and variation in the levels of cell markers. (A) Effect of tPA deficiency on antigen presentation, as indicated by MOG35-55-specific T-cell proliferation. Lymphocyte proliferation was assessed by [3H] thymidine incorporation using APCs from wt EAE or tPA−/− EAE mice in the presence of MOG35-55. (B) Splenocytes derived from tPA−/− and wt EAE mice were analyzed for expression of surface markers by FACS analysis, as described in the methods (n = 6 in each group) (*p < 0.05, **p < 0.001).

Table 2 Cell markers in naïve tPA−/− and wt mice.

CD4 CD8 CD3 CCR5 CD25 CD19 CD11b

Naïve % of cells

tPA−/− % of cells

20 ± 0.8 13.6 ± 0.7 32.6 ± 2.1 4.5 ± 0.4 3.2 ± 0.4 47.6 ± 4.5 5.4 ± 0.05

18.6 ± 2.0 12.5 ± 0.8 31.6 ± 1.1 4.2 ± 1.0 2.3 ± 0.1 43.3 ± 6.2 3.5 ± 0.3

Splenocytes derived from naïve tPA−/− and wt mice, analyzed for expression of surface markers by FACS analysis, as described in the methods. Data presented are the mean ± SE values determined from three separate experiments.

Secretion of the pro-inflammatory cytokines IFNγ and IL-17 was decreased by 93% and 62% respectively by treatment with tPA-S(481)A (Fig. 6D), and following treatment with PAI-1-dp a reduction by 84% and 77% respectively (p = 0.001) (Fig. 6D). It should be noted that there was no statistical significance when comparing the cellular reactivity between the two modes of treatment (tPA-S(481)A versus PAI-I dp). Further, secretion of the anti-inflammatory cytokine IL-10 was increased by 12 times (Fig. 6C). We also found a significant reduction in the mRNA expression of the transcription factor RORγt (a marker for Th17 cells) as well as T-bet (a marker for Th1 cells) (Fig. 6E).

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Fig. 5. Reduction in the number of Treg cells in tPA−/− mice. (A) Splenocytes derived from EAE-induced tPA−/− and wt mice stained for the Treg markers CD4 (X axis) and CD25 (Y axis) (upper panel) and FoxP3 (lower panel), as analyzed by FACS. CD4/CD25-double positive cells were gated and assessed for FoxP3 expression. The percentage of Tregs was calculated as the percentage of cells expressing all three markers. (B) The results are shown as the percentage of CD4/CD25/Foxp3-positive Treg cells derived from the spleen of wt and tPA−/− mice in the naïve state and after EAE induction. (C) FoxP3 mRNA expression level. (D) Splenic T-regs are upregulated following PAI-1 treatment. The findings are shown as the percentage of Treg-positive cells. The data presented are the mean ± SE values from three separate experiments (*p < 0.05).

inflammatory. The former is related to the producing of adjusted innate and adaptive immune responses, whereas the latter is related to prevention of extracellular fibrin deposition [36].

The immunopathogenesis of EAE involves specific CD4 + T cells [37], and their activation is determined by their interactions with APCs, of which the most prominent are CD11b macrophages and CD11c-

Fig. 6. Reduction in T-cell reactivity after treatment of wt EAE mice with S(481)A protein or PAI-1 peptide. (A) Proliferation of splenocytes from placebo- treated or S (481)A-treated EAE mice as determined by [3H] thymidine incorporation in the presence of MOG35-55. (B) Proliferation of splenocytes from placebo- treated or PAI-I d.p EAE mice as determined by [3H] thymidine incorporation in the presence of MOG35-55. (C) Effect of S(481)A treatment on cytokine secretion. Lymphocytes from the spleens of placebo- and S(481)A-treated EAE mice were stimulated with MOG35-55 or anti-CD3 antibody, The culture media were collected for IL-10 (C) or IFN-g and IL-17 (D) assay. The secreted cytokines were measured by ELISA. (E) Transcription factors mRNA expression level, following activation with MOG35-55. The results shown are the mean ± SE values from three different experiments (*p < 0.05, **p < 0.001). 7

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expressing dendritic cells. Thus, the contradictory finding could be explained by intrinsic defects present in T-cells, APCs or both cell types from the tPA−/− mice. In fact, bidirectional communication links the PA system and inflammatory cytokine networks. On the one hand, inflammatory mediators can modulate the expression of the PA system components [38–42], while on the other hand, it was shown that uPA is a modulator of immune and inflammatory responses [43]. Our findings indicated that mice lacking tPA present with more severe disease as well as the marked effect of tPA deficiency on lymphocyte activation in our model, and the deleterious clinical outcome, can be explained by the inability of the tPA−/− mice to mount a protective immune response. In agreement with our findings, Gyetko et al. [44], who studied the ability of uPA−/− mice to activate a protective host defense during infection with Cryptococcus neoformans, found that the Th1 responses failed to develop, the antigen-specific lymphocyte reaction was blunted, and the impaired immune response of the mice was lethal [44]. Additionally, in our study, tPA−/− EAE mice had poorer antigen presentation capacity and a lower percentage of CD11b APCs. This may be attributable to a defect in the migration of the macrophages from the site of inflammation, as this is essential for the resolution of acute inflammation and the initiation of adaptive immunity [45]. That is, the inability of macrophages to migrate properly may affect T-cell reactivity, which in turn may have a negative effect on the resolution of inflammation and disease severity. CeC chemokine receptor 5 (CCR5) has been implicated in immune cell migration and cytokine release in the CNS. Gu et al. [46] reported that CCR5−/− mice develop less severe EAE than wt mice. Additionally, patients with MS exhibit a higher percentage of circulating CCR5 + cells than controls, and an increase in the number of these cells is associated with disease severity. In the present study, unexpectedly, we found that mice lacking tPA with aggravation of EAE exhibited reduced CCR5 levels. However, this finding fits in with the results that show decreased lymphocyte reactivity and cytokine secretion. Since T-regulatory cells are known to play a pivotal role in EAE and MS, they were evaluated in the present study. We found a reduction in T regulatory cells, which can also explain the more severe disease in the tPA−/− mice. Further, the reduction in Tregs was observed in the naïve state, too, and it was amplified after induction of EAE. These results are in agreement with our previous results which show that tPA−/− mice develop more severe experimental autoimmune myasthenia gravis, with less T-regulatory cells, less lymphocyte activity and fewer APCs (dendritic cells and macrophages). As expected, following treatment with tPA S(481)A, the immunological response was reduced, T-cell activation was reduced, and pro-inflammatory cytokine secretion was also reduced, while secretion of anti-inflammatory cytokines was increased.

Funding The study was supported by the European Union grant no. 242210 to T.B. and partly by a grant from Israel Ministry of Health Chief Scientist fund. Ethics approval The study is approved by the ethic committee of the Hebrew University. NIH approval number: OPRR-A01-5011, MD-1413867-5. Author declaration Tehila Mizrachi performed the animal studies and the cell culture and phatlogical studies and was involved in writing all the versions of the manuscript. Devora Gur-Wahnon was involved in the cell culture experiments and evaluation. Talma Brenner and Abd Al-Roof Higazi were involved in study design and ideas formulation. Talma Brenner involved in writing the various versions of the manuscript and acquisition of the financial support for the project. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cellimm.2020.104040. References [1] C.S. Constantinescu, N. Farooqi, K. O'Brien, B. Gran, Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS), Br. J. Pharmacol. 164 (2011) 1079–1106. [2] L.M. Matrisian, Metalloproteinases and their inhibitors in matrix remodeling, Trends Genet. 6 (1990) 121–125. [3] H. Birkedal-Hansen, Proteolytic remodeling of extracellular matrix, Curr. Opin. Cell Biol. 7 (1995) 728–735. [4] P.A. Andreasen, L. Kjoller, L. Christensen, M.J. Duffy, The urokinase-type plasminogen activator system in cancer metastasis: a review, Int. J. Cancer 72 (1997) 1–22. [5] J.L. Jones, R.A. Walker, Control of matrix metalloproteinase activity in cancer, J. Pathol. 183 (1997) 377–379. [6] J.P. Irigoyen, P. Munoz-Canoves, L. Montero, M. Koziczak, Y. Nagamine, The plasminogen activator system: biology and regulation, Cell. Mol. Life Sci. 56 (1999) 104–132. [7] T. Teesalu, A. Kulla, T. Asser, M. Koskiniemi, A. Vaheri, Tissue plasminogen activator as a key effector in neurobiology and neuropathology, Biochem. Soc. Trans. 30 (2002) 183–189. [8] J. Felez, C.J. Chanquia, E.G. Levin, L.A. Miles, E.F. Plow, Binding of tissue plasminogen activator to human monocytes and monocytoid cells, Blood 78 (1991) 2318–2327. [9] T. Teesalu, A.E. Hinkkanen, A. Vaheri, Coordinated induction of extracellular proteolysis systems during experimental autoimmune encephalomyelitis in mice, Am. J. Pathol. 159 (2001) 2227–2237. [10] E. East, D. Baker, G. Pryce, H.R. Lijnen, M.L. Cuzner, D. Gveric, A role for the plasminogen activator system in inflammation and neurodegeneration in the central nervous system during experimental allergic encephalomyelitis, Am. J. Pathol. 167 (2005) 545–554. [11] D. Gur-Wahnon, T. Mizrachi, F.Y. Maaravi-Pinto, A. Lourbopoulos, N. Grigoriadis, A.A. Higazi, T. Brenner, The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention, J. Neuroinflamm. 10 (2013) 124. [12] Y.H. Yang, P. Carmeliet, J.A. Hamilton, Tissue-type plasminogen activator deficiency exacerbates arthritis, J. Immunol. 167 (2001) 1047–1052. [13] M. Salazar-Paramo, I. Garcia de la Torre, M.J. Fritzler, S. Loyau, E. Angles-Cano, Antibodies to fibrin-bound tissue-type plasminogen activator in systemic lupus erythematosus are associated with Raynaud's phenomenon and thrombosis, Lupus 5 (1996) 275–278. [14] M. Ieko, Antiphospholipid antibodies and thrombosis: the putative mechanisms of hypercoagulable state in patients with anticardiolipin antibody, Rinsho Byori 48 (2000) 293–300. [15] T. Nassar, S. Akkawi, A. Shina, A. Haj-Yehia, K. Bdeir, M. Tarshis, S.N. Heyman, A.A. Higazi, In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone, Blood 103 (2004) 897–902. [16] M.J. Cuadrado, M.A. Khamashta, The anti-phospholipid antibody syndrome (Hughes syndrome): therapeutic aspects, Baillieres Best Pract. Res. Clin. Rheumatol.

5. Conclusions Our findings indicate that tPA plays an immunomodulatory role in the pathogenesis of EAE. Further, neutralization of the endogenous inhibitor of tPA or administration of tPA variant proteins ameliorated the disease. Thus, tPA may have potential for the immunomodulation of neuroinflammation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank Mrs Camille Sicsic for her skillful assistance. 8

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