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Application of a triple‐helical peptide inhibitor of MMP‐2/MMP‐9 to examine T‐cell activation in experimental autoimmune encephalomyelitis

Application of a triple‐helical peptide inhibitor of MMP‐2/MMP‐9 to examine T‐cell activation in... INTRODUCTIONThe active stage of multiple sclerosis (MS) is characterized by the breakdown of the blood–brain barrier (BBB), perivascular infiltration of inflammatory auto‐reactive T‐cells in the central nervous system (CNS), and demyelination. Matrix metalloproteinases (MMPs) are responsible for turnover and remodeling of the extracellular matrix (ECM) and CNS. The normal CNS contains low levels of MMPs, but several MMPs are upregulated in neurological disorders of the CNS, including MS.[1] MMPs are assumed to act as effector molecules in the (a) disruption of the BBB, including targeting of tight junction proteins (occludin, ZO‐1, and claudin‐5), basal lamina proteins (fibronectin, laminin, collagen, and heparan sulfate), chemokines, and growth factors,[2–5] (b) invasion of inflammatory cells into the CNS parenchyma,[2,3,6,7] (c) degradation of myelin‐associated glycoprotein (MAG) and myelin basic protein (MBP),[3,8] and (d) toxicity to axons and neurons.[1,9,10] MAG and MBP fragments are neuroantigens associated with the sensitization/activation of immune cells such as T‐cells. Sensitized T‐cells in the periphery are believed to cross the BBB and initiate a second wave of immune responses that activate microglia cells/macrophages and astrocytes to release proinflammatory cytokines and MMPs, contributing to further myelin degradation and axonal loss.[3,10] Imaging of MMP activity using a positron emission tomography‐compatible MMP inhibitor defined both relapsing and progressive active MS.[7]The application of synthetic MMP inhibitors in experimental autoimmune encephalomyelitis (EAE), a widely accepted animal model of MS, has demonstrated reduced clinical severity following administration after disease induction or onset of disease symptoms.[11–14] Effects were apparently mediated through restoration of the damaged BBB in the inflammatory stage of the disease[11] or reduction of leukocyte entry into the CNS,[12] although a lack of demyelination was observed in some cases.[13] A deficiency of tissue inhibitor of metalloproteinase (TIMP)‐1, an endogenous inhibitor of MMPs, in EAE mice results in persistent macrophage activation, myelin disruption, and increased disease severity.[10]Numerous MMPs have been implicated in MS. Of particular interest is the observation that MMP‐9 levels are elevated in MS patient serum, plasma, and cerebrospinal fluid, particularly in patients with active disease.[3,7,9,15–19] In addition, an increase in the MMP‐2:TIMP‐2 ratio marks chronic progression in MS while a high MMP‐9:TIMP‐1 ratio characterizes relapsing remitting MS.[1] Numerous studies have correlated increased MMP‐9 activity with the breakdown of the BBB, possibly through the degradation of tight junction proteins.[20–27] MMP‐9 derived from immune cells is critical for leukocyte penetration into the CNS parenchyma.[7] MMP‐9 inhibition reduces significantly the ability of naïve T‐cells to respond to stimulation.[28] MMP‐2, MMP‐9, MMP‐10, and MT6‐MMP cleave MBP efficiently.[29] Loss of both MMP‐2 and MMP‐9 results in the inability to induce EAE in mice,[30] while loss of MMP‐9 alone leaves young (but not older) mice significantly less susceptible to EAE initiation.[1,31] Hydrolysis of β‐dystroglycan by MMP‐2 and MMP‐9 promotes leukocyte penetration of the parenchymal basement membrane during EAE.[30] In a related study, a 7 day treatment of MMP‐2/MMP‐9/MMP‐14 inhibitor SB‐3CT[32,33] in a mouse model of ischemia was found to be neuroprotective.[34,35]The use of MMP inhibitors for MS treatment has precedent. Doxycycline and minocycline, which are broad‐spectrum MMP inhibitors with IC50 values in the range of 2–50 and 100–300 μM, respectively,[36] have been utilized in clinical trials of MS patients. For example, treatment of 16 relapsing–remitting MS patients with a doxycycline/interferon combination for 4 months (ClinicalTrials.gov Identifier NCT00246324) reduced brain lesions and MMP‐9 serum levels, improved Expanded Disability Status Scale values, and was considered safe and well‐tolerated.[2,37] In similar fashion, minocycline treatment of relapsing–remitting MS patients resulted in no new active lesions after 1 month with detection up to 6 months for 10 patients[38] while combination treatment of minocycline with glatiramer acetate in 44 patients showed reduced number of lesions by 63%–65% and lowered risk of relapse after 8–9 months compared with glatiramer treatment alone.[39] Additional clinical trials using minocycline for MS treatment are reportedly upcoming.[4] Future correlation of MMP activity with MS subtypes[40] may provide insight into patient‐specific treatment strategies.Our laboratory has developed selective, high affinity MMP inhibitors modeled on the triple‐helical structure of collagen.[14,41–46] These triple‐helical peptide inhibitors (THPIs) utilized phosphinate analogs of Gly‐Leu, Gly‐Ile, and Gly‐Val to bind to the MMP active site Zn(II).[41,42,44] α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI was selective for MMP‐2 and MMP‐9, with Ki values of 190 and 91 nM, respectively, at 25°C, and 2 and 1 nM, respectively, at 37°C.[14] When administered intraperitoneally at a dose of 12.43 μg/mouse per day starting from day 7, THPI treatment reduced the clinical severity of EAE from day 12 on.[14] The THPI treated EAE mice on average had less weight loss per day and increased their weight before the control group.[14] However, the mechanism by which the THPI improved clinical outcomes in EAE mice was unknown. In the present study we used the α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI to assess its efficacy in mitigating key immune cell mediators of MS, such as CD4+ T‐cells, by measuring cytokine secretion following THPI treatment. T‐cells, and specifically CD4+ T‐cells from relapsing–remitting MS patients, have been shown to produce MMP‐9 and utilize it to promote leukocyte migration into the CNS.[7,13,28,47–49] Anti‐CD3 stimulation of CD4+ T‐cells induced MMP‐9 expression and increased protein production and release.[28] Herein we assessed the in vitro impact of the THPI on the responses associated with CD4+ T‐cell activation, such as cellular proliferation and the secretion of the key cytokines, such as interleukin (IL)‐2, tumor necrosis factor α (TNF‐α), and interferon γ (IFN‐γ). We then applied the THPI in vivo in the EAE model to evaluate pre‐clinical response and splenocyte proliferation and production of cytokines.MATERIALS AND METHODSPeptide synthesis and purificationThe α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI [sequence (Gly‐Pro‐Hyp)4‐Gly‐mep‐Flp‐Gly‐Pro‐Pro‐GlyΨ{PO2H‐CH2}Val‐Val‐Gly‐Glu‐Gln‐Gly‐Glu‐Gln‐Gly‐Pro‐Pro‐Gly‐mep‐Flp‐(Gly‐Pro‐Hyp)4‐NH2, where Hyp = 4‐hydroxy‐L‐proline, mep = (2S,4R)‐4‐methylproline, and Flp = (2S,4R)‐4‐fluoroproline] was synthesized and characterized as previously described.[14]In vitro cell cultureCD4+ T‐cells were purified from spleen or lymph nodes of C57/Bl6 naïve mice via positive selection (CD4+ T‐cell isolation kit, Miltenyi Biotech) according to manufacturer's instructions. Purity was confirmed to be greater than 95%. CD4+ T‐cells were then cultured at a density of 1 × 106/mL in cRPMI, consisting of Roswell Park Memorial Institute (RPMI) 1640 plus 10% fetal bovine serum and 100 u/mL Pen‐Strep. To assess the impact of the THPI on CD4+ T‐cells, T‐cells were cultured for 24 h in the presence or absence of THPI followed by stimulation of the TCR using CD3/CD28 mAb Dynabeads (Invitrogen) as per manufacturer's instructions. In the case of splenocytes, the same culture conditions were utilized using a single cell suspension of the splenocytes from EAE or control mice. The splenocytes were treated in vitro with 10 μM THPI for 24 h, rinsed twice with RPMI, and stimulated with 50 μg/mL of MOG35−55 peptide for 96 h.Enzyme‐linked immunosorbent assayCell‐free culture supernatant from PBS or inhibitor treated CD4+ T‐cells or splenocytes were collected at 96 h and used for protein analysis of IL‐2, TNF‐α, IFN‐γ, IL‐6, and IL‐10 via enzyme‐linked immunosorbent assay (ELISA; all kits from BD Biosciences). The ELISA assays utilized mouse mAbs against recombinant IL‐2, IL‐6, IL‐10, and IFN‐γ and a purified mouse Ab against recombinant TNF‐α. Absorbance at λ = 450 nm with a wavelength correction at λ = 570 nm was measured with an H1 Biotek plate reader. The optical density values of samples recorded was converted to picogram using a standard curve obtained using known quantities of recombinant cytokines according to manufacturer's instructions.Toxicity assayCell toxicity was evaluated using the CellTox Green Cytotoxicity Assay (Promega) according to manufacturer's instructions. Fluorescence was recorded at λexcitation = 490 nm and λemission = 520 nm using an H1 Biotek plate reader.Proliferation assayProliferation was assessed using the CellTiter 96 AQueous proliferation assay (Promega) according to manufacturer's instructions. Absorbance at λ = 490 nm was recorded using an H1 Biotek plate reader.Murine model of MSAll procedures involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (protocol # TPI 12‐11 FL and # A18‐04, IBC # 2018‐269). Complete Freund's Adjuvant was produced by emulsifying Incomplete Freund's Adjuvant (Thermo Scientific) and M. tuberculosis H37Ra (Becton Dickinson, Franklin Lakes, NJ). MOG35−55 peptide (Met‐Glu‐Val‐Gly‐Trp‐Tyr‐Arg‐Ser‐Pro‐Phe‐Ser‐Arg‐Val‐Val‐His‐Leu‐Tyr‐Arg‐Asn‐Gly‐Lys‐OH) was synthesized using standard Fmoc solid‐phase chemistry[50] and RP‐HPLC purified to >90%. EAE was induced in female C57BL/6J mice (Jackson Laboratory; 10–12 weeks of age) by subcutaneously inoculating an emulsification of Complete Freund's Adjuvant and the MOG35−55 peptide (200 μg/mouse) on Day 0. Pertussis Toxin (Hooke's Laboratory, 320 ng) was introduced via intra‐peritoneal injection 3 and 24 h later. The control group received PBS/animal/day, while the treated group received 0.03 μg α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI/animal/day from day 7 by intra‐peritoneal injection. Disease severity (scored based on the Hooke's Laboratory scoring guide [www.hookeslab.com/protocols/eaeAI_C57BL6.html]) and weight change were recorded from Day 5 until sacrifice.RESULTSIn MS and its animal model, EAE, T‐cells become sensitized to neuroantigens by the concurrent stimulation of the T‐cell receptor (TCR) and its costimulatory molecule, CD28, in the presence of the neuroantigen. In vitro this process is effectively reproduced by the administration of CD3/CD28 antibodies.[28,51] Thus, to examine the effect of the THPI in T‐cell functionality, CD4+ T‐cells were enriched from the splenocytes of C57/BL6 mice, treated in the presence or absence of THPI, and stimulated with anti‐CD3/CD28 mAbs.THPI effects on proliferation and cytokine production of naïve T‐cellsOnce CD4+ T‐cells experience antigenic stimulation, subsets of these lymphocytes respond by proliferating and producing pro‐proliferation cytokines such as IL‐2 as well as the effector cytokines IFN‐γ and TNF‐α. Proliferation is thus a hallmark of a robust stimulation that helps to magnify the immune response and is aided by the autocrine actions of IL‐2. Thus, we sought to determine what impact THPI treatment would have on CD4+ T‐cell stimulation. THPIs need approximately 24 h incubation to adequately assume triple‐helical structure[52]; and we allowed an additional 24 h time frame once added to cells to allow for the reformation of the triple‐helical structure in the advent of any potential disruption associated with the cell culture process. CD4+ T‐cell toxicity due to the THPI was assessed and not observed (data not shown). A reduction trend in CD4+ T‐cell proliferation was observed at [THPI] = 25 μM (Figure 1).1FIGUREPurified CD4+ T‐cells were magnetically separated from the splenocytes of C57/BL6 naïve mice and pooled (n = 5) due to limited availability of samples. CD4+ T‐cells were then cultured with the THPI for 24 h, rinsed twice with RPMI‐1640, and then stimulated with CD3/CD28 mAbs in cRPMI for 72 h. Data is representative of two independent experimentsBased on the proliferation results, the impact of the THPI on cytokine production associated with T‐cell activation was determined after a 24 h incubation in culture and at a THPI concentration of 15 μM. IL‐2, which promotes CD4+ T‐cell proliferation upon CD3/CD28 activation,[53] was initially examined. There was a marked reduction in IL‐2 protein production (Figure 2 upper left panel) upon THPI treatment. The results indicated that the THPI hampered the ability of naïve CD4+ T‐cells to proliferate in response to antigenic stimulation.2FIGUREPurified CD4+ T‐cells were magnetically separated from the splenocytes of C57/BL6 naïve mice and pooled (n = 5) due to limited availability of samples. CD4+ T‐cells were then cultured with THPI for 24 h, rinsed twice with RPMI‐1640, and then stimulated with CD3/CD28 mAbs in cRPMI for 72 h. Cell free supernatant was collected and used to determine protein levels of IL‐2 (upper left), IFN‐γ (upper right) and TNF‐α (bottom) by ELISA. Data is representative of three independent experiments. T‐cells cultured with THPI for 24 h prior to 72 h CD3/CD28 stimulation significantly reduced production of IL‐2 (****p < 0.0001), IFN‐γ (****p < 0.0001), and TNF‐α (***p < 0.001). Statistical analysis was performed using a Student's t‐testTHPI treatment was next evaluated for effects on CD4+ T‐cell ability to produce effector cytokines that activate and/or stimulate other immune cells. In this case, the production of cytokines IFN‐γ and TNF‐α was quantified. IFN‐γ is an activator for the T‐cell effector phase and may directly induce apoptosis[54,55] while TNF‐α enhances CD4+ T‐cell proliferation and increases cytokine production.[56] THPI treatment of CD4+ T‐cells prior to CD3/CD28 stimulation reduced IFN‐γ and TNF‐α protein levels (Figure 2 upper right and bottom panels). These results suggested that the THPI can suppress CD4+ T‐cell effector function by reducing the production and/or secretion of IFN‐γ and TNF‐α.THPI treatment of EAE miceEAE is a well‐established murine model of MS, where the most widely used EAE model is the chronic progressive model in which C57BL/6J mice are immunized with myelin oligodendrocyte glycoprotein (MOG)‐derived peptide fragments.[57,58] EAE was induced herein with the MOG35−55 peptide.[13] Days 7–10 approximate the time of T‐cell activation and migration across the BBB which manifests as disease onset. Approximately 3–5 days after initial onset maximum clinical severity is observed.In contrast with our prior study,[14] we presently utilized the THPI at a dose (0.03 μg) more representative of practical application. Clinical symptoms of EAE appeared at day 10 and continued until the end of the experiment (day 20) (Figure 3, left panel). Untreated animals also exhibited weight loss (Figure 3, right panel). THPI treatment reduced the clinical severity of EAE from day 12 on (Figure 3, left panel). The THPI treated mice on average had less weight loss per day and increased their weight before the control group (Figure 3, right panel). Increased clinical severity and weight loss are hallmarks of EAE.3FIGUREResults of 0.03 μg α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI/day treatment of EAE mice. (left) Clinical score of THPI treated (gray) and nontreated (black) mice. (right) Weight compared with starting weight of THPI treated (gray) and nontreated (black) mice. n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was performed using a two‐tailed Student's t‐testSplenocytes were isolated from THPI‐treated and untreated EAE mice at day 21. Splenocytes from THPI‐treated EAE mice were found to have reduced proliferation compared with untreated EAE mice (Figure 4, left panel). To determine if the reduced proliferation was due to cell death, isolated splenocytes were evaluated for toxicity. There was no significant difference in the degree of cytotoxicity between THPI‐treated and untreated EAE mice (Figure 4, right panel).4FIGUREAnalysis of splenocyte (left) proliferation and (right) cytotoxicity on day 21 following 0.03 μg α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI/day treatment of EAE mice. In vivo THPI administration reduced splenocyte proliferation in response to antigen, but the THPI was not toxic to splenocytes, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was performed by using a Student's t‐testTHPI effects on proliferation and cytokine production of splenocytes isolated from EAE miceT‐cells from diseased mice are known to have a different baseline cellular activity as compared with T‐cells from naïve mice who have not experienced any pathological conditions. We next assessed if THPI treatment would have similar effects on not just T‐cells from diseased mice, but on the overall immune response of which CD4+ T‐cells play a key role in orchestrating. Thus, splenocytes from EAE mice were isolated for an in vitro recall experiment.Following isolation, the splenocytes were treated with 10 μM of THPI for 24 h and either stimulated with 50 μg/mL of MOG35−55 peptide or not stimulated for 96 h. In addition to IL‐2 the cytokines IL‐10 and IL‐6 were monitored. IL‐10 is an anti‐inflammatory cytokine[59] while IL‐6 enhances CD4+ T‐cell proliferation and differentiates CD4+ T‐cells into subsets (see Section 4).[60,61] Production of IL‐2, IL‐10, and IL‐6 after 36 h was quantified. The unstimulated splenocytes had little (first two columns of Figure 5 upper left and upper right panels) to no (first two columns of Figure 5 bottom panel) cytokine production. MOG stimulated splenocytes (the third columns of Figure 5) produced significant amounts of IL‐2, IL‐10, and IL‐6. In turn, THPI treatment significantly reduced production of IL‐2 (fourth column of Figure 4 upper left panel), IL‐10 (fourth column of Figure 5 upper right panel), and IL‐6 (fourth column of Figure 5 bottom panel).5FIGURESplenocytes isolated from untreated EAE animals were treated with and without THPI for 24 h and stimulated with and without MOG33−35 for 96 h. Cell free supernatant was used in an ELISA to determine protein levels. The production of (top left) IL‐2, (top right) IL‐10, and (bottom) IL‐6 was significantly reduced in stimulated cells, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Statistical analysis was performed using a Student's t‐testSplenocytes were treated as described above and effector cytokine levels quantified. The unstimulated splenocytes did not produce significant amounts of IFN‐γ (first two columns of Figure 6 left panel); on the other hand, TNF‐α production from unstimulated splenocytes was evident and was reduced by THPI treatment (first two columns of Figure 6 right panel). The MOG stimulated splenocytes produced IFN‐γ (third column of Figure 6 left panel) and increased levels of TNF‐α compared with unstimulated splenocytes (first and third columns of Figure 6 right panel). IFN‐γ and TNF‐α levels were significantly reduced following THPI treatment (fourth column of Figure 6 both panels). The results indicated that THPI treatment could reduce the effector response of immune cells during EAE, thus mitigating disease severity.6FIGURESplenocytes isolated from untreated EAE animals were treated with and without THPI for 24 h and stimulated with and without MOG33−35 for 96 h. Cell free supernatant was used in an ELISA to determine (left) IFN‐γ and (right) TNF‐α protein levels. The production of IFN‐γ and TNF‐α was reduced, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was performed using a Student's t‐testDISCUSSIONThe present study was designed to initially evaluate if MMP‐2/MMP‐9 activity of T‐cells potentially contributed to EAE pathology. As a first step, a homogenous population of murine CD4+ T‐cells obtained from the spleen of naïve mice was pre‐treated with an MMP‐2/MMP‐9‐targeting THPI, followed by stimulation of the TCR using CD3/CD28 antibodies. A modest suppression of T‐cell proliferation at [THPI] = 25 μM was observed without cellular toxicity. We allowed for a 24 h time frame for the triple‐helix to form in the dilutions, and then added them directly to cells. The THPI concentration of 10–15 μM assured a well‐formed triple‐helix and the potential for an initial effect on CD4+ T‐cell proliferation.For the EAE studies the THPI dose used, in theory, approximates the α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI Ki for MMP‐2 and MMP‐9[14] based on the following calculation. With a molecular weight of 12.4 kDa, one 0.03 μg dosage of α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI corresponds to 2.4 × 10−12 mol. Mice have ~58.5 mL of blood per kg of bodyweight. Hence, a mouse weighing 25 g would have a total blood volume of ~58.5 mL/kg × 0.025 kg = 1.46 mL. Consequently, the blood concentration of α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI would reach 2.4 × 10−12 mol/1.46 × 10−3 L = 1.64 nM.THPI treatment of CD3/CD28 stimulated CD4+ T‐cells resulted in the reduction of the (a) initial proliferation of CD4+ T‐cells, (b) production of the proliferative cytokine IL‐2, and (c) production of effector cytokines IFN‐γ and TNF‐α. These indicators of T‐cell activity were subsequently utilized to evaluate the activity of the THPI in the EAE model of MS. The THPI reduced the clinical severity of EAE and splenocytes isolated from THPI‐treated EAE mice had reduced proliferation. In this latter case proliferation (Figure 4, left panel) is due to antigen‐specific (MOG35−55 peptide) activation, whereas in the former case proliferation (Figure 1) is due to general T‐cell stimulation. The two cases represent immunologically distinct circumstances which accounts for the modest effect observed with THPI inhibition of general T‐cell stimulation and the significant effect observed with THPI inhibition of antigen‐specific splenocyte activation.To determine if the THPI effects on CD4+ T‐cells was recapitulated in EAE mice, splenocytes were isolated, stimulated with auto‐antigen (MOG35−55), and treated with the THPI. Splenocytes capture a significant range of immune cells, 19%–22% CD4+ T‐cells, ~18% CD8+ T‐cells, ~2% CD4+ regulatory T‐cells, 18%–21% B cells, ~3% natural killer cells, ~2% immature myeloid cells, 2%–4% myeloid dendritic cells, 4%–5% macrophages, and 2%–3% neutrophils.[62] Similar effects were observed with splenocytes isolated from EAE mice compared with CD4+ T‐cells isolated from naïve mice. Of the cytokines examined, IL‐2 allows for a robust immune response via T‐cell proliferation, IL‐10 can downwardly regulate the immune response, and IL‐6 can shape the immune response in EAE to a highly aggressive form. As the immune response moves into the effector phase, cytokines such as IFN‐γ and TNF‐α become important for the destruction of target cells and recruitment of other immune cells. These data also suggest that other immune cells present in the splenocytes are sensitive to the effects of the THPI.The innate arm of the immune system (macrophages, dendritic cells, neutrophils) is typically the first to encounter an immunogen. In MS these cells phagocytize neuroantigens such as MBP and MOG and break them down into small fragments, for presentation to the TCR. This process sensitizes the T‐cells to the neuroantigen and establishes neuronal cells as target cells. In MS and EAE, T‐helper 1 (Th1) and Th17 cell subsets are the key mediators of pathological conditions.[63] MMP‐9 generates immunogenic epitopes that induce Th1 and Th17 cell subsets.[64]Naïve T‐cells and activated Th1 and Th2 cells produce cytokines such as IL‐2, IL‐6, IL‐10, and IL‐12, associated with the sensitization phase of EAE,[65–69] while activated Th1 and Th17 cells produce cytokines such as IL‐17, IFN‐γ, and TNF‐α, associated with the effector phase of EAE.[57] These cytokines influence the magnitude of the immune response in EAE and MS.[70,71] For example, TNF‐α is associated with successful stimulation of the TCR pathway and key mediation of immune cell assault on the CNS during EAE and MS. Thus, one way in which the THPI mitigated EAE clinical outcomes is by suppression of TNF‐α production (Figure 6).In the present study the treatment of activated CD4+ T‐cells with the MMP‐2/MMP‐9 selective THPI resulted in decreased cell proliferation and production of IL‐2, IFN‐γ, and TNF‐α (Figures 1 and 2). These results are consistent with prior reports. Treatment of T‐cells with minocycline, a broad‐spectrum MMP inhibitor (see below), resulted in decreased cell proliferation and production of IL‐2, IFN‐γ, and TNF‐α.[72] Treatment of anti‐CD3‐stimulated CD4+ T‐cells with MMP‐2/MMP‐9/MMP‐14 inhibitor SB3CT resulted in decreased cell proliferation, IL‐2 production, and CD25 expression.[28] Anti‐CD3‐stimulated CD4+ T‐cells isolated from MMP‐9 deficient mice (MMP9−/−) showed an 80%–85% decrease in proliferation, reduced IL‐2 production, increased intracellular calcium flux, and reduced CD25 and CD69 expression compared with CD4+ T‐cells from wild‐type mice.[28] In addition, anti‐CD3‐stimulated MMP9−/− CD4+ T‐cells showed increased CD62L, CTLA‐4, and CD45RO expression compared with CD4+ T‐cells from wild‐type mice.[28]A recent study examined the effects of naïve CD4+ T‐cell stimulation by CD3 and CD28 antibodies in the presence of collagen‐derived peptides Pro‐Hyp and Hyp‐Gly.[73] These peptides could presumably result from the action of MMP‐9, amongst other proteases, on collagen. Collagen peptide ingestion resulted in increased production of IL‐10 and IFN‐γ by splenocytes, but not IL‐2, IL‐6, or TNF, suggesting a shift towards a Th1‐dominant state.[73] The action of the peptides was through enhancement of STAT1 signaling. The effects observed in our study are different, in that the production of both IFN‐γ and TNF was suppressed by THPI treatment, and thus indicating that the presumed MMP‐2/MMP‐9 action herein was not through the same pathway as the degradation of ECM molecules.Although not identified here, one can consider the possible MMP‐2/MMP‐9 substrate(s) that result in modulation of T‐cell behaviors. The prior study on inhibition of T‐cell proliferation by SB3CT did not identify the MMP substrate but suggested that it was intracellular.[28] Utilizing a semi‐selective THPI with high affinity for MMP‐9, MMP‐9 was found to cleave citrate synthase in vitro and in vivo, generating a ~20 kDa fragment.[74] 5′‐adenosine monophosphate‐activated protein kinase α subunit has been identified as an MMP‐9 substrate in myeloid cells.[75] Both citrate synthase and 5′‐adenosine monophosphate‐activated protein kinase are key players in cellular energy homeostasis. Prior studies have identified numerous potential intracellular substrates for MMP‐2 and MMP‐9, including enzymes involved in carbohydrate metabolism and protein biosynthesis.[76] Alternatively, MMP‐2/MMP‐9 could be acting on T‐cells via processing cell surface biomolecules. It has been proposed that the major role of MMPs is modulating inflammatory and immune processes via processing of signaling molecules.[4,19,77] Along these lines, activation of T‐cells and subsequent production of IFN‐γ and IL‐2 was previously shown to be modulated by ADAM10 or ADAM17 cleavage of lymphocyte activation gene‐3 (CD223) from the cell surface.[78] We have applied machine learning in combination with RNA sequencing to uncover potential MMP‐2/MMP‐9 substrates in T‐cells[79] and will pursue this avenue to identify relevant MMP‐2/MMP‐9 substrates in EAE. The fact that all cytokines tested in the present study were reduced suggests that the MMP‐2/MMP‐9 substrate processing inhibited by the THPI impacts the cellular machinery in broad manner. A number of cytokines are known substrates of MMP‐2 and/or MMP‐9, such as interferon‐β (used for the treatment of MS), IL‐2, and monocyte chemoattractant protein‐3, and are functionally altered by the action of these proteinases.[80–82] Thus, of particular relevance here, in addition to indirect effects on adaptive immune processes, the inhibition of MMP‐9 can have a direct effect on the bioavailability of IL‐2.[82]As previously discussed,[14,83] THPIs have several favorable properties that facilitate their use as probes of enzyme activity and imaging agents in vivo, including the excellent stability of triple‐helical peptides in vivo[84–86] and the efficacy of MMP inhibiting phosphinic peptides in vivo.[87] The α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI structure facilitates binding of the inhibitor to the targeted MMPs while minimizing proteolytic degradation. Although MMP‐9 selective, inhibitory antibodies and antibody fragments have been described,[83,88] their application for neurological disorders is limited based on the inefficiency of antibodies to cross an intact BBB although they may cross a compromised BBB during active disease. One goal of using MMP inhibitors is to rapidly restore a compromised BBB (by minimizing proteolysis of tight junction proteins such as claudin‐5 and occludin[89]). Restoration of the intact BBB should allow small molecules to more efficiently enter the brain and inhibit additional detrimental processes in the CNS. The effectiveness of the MMP‐2/MMP‐9 α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI in the EAE mouse model indicated that this inhibitor could cross the BBB. Importantly, the inhibitory activities of the first generation THPI [C6‐α1(V)GlyΨ{PO2H‐CH2}Val THPI] were similar for full‐length (82 kDa) MMP‐9 and TIMP‐1 resistant (65 kDa) MMP‐9[42] and the TIMP‐1 resistant MMP‐9 appears to be the predominant enzyme form contributing to MS.[90–92]At the onset of EAE, MMP‐9 has a disease‐promoting effect, as evidenced by knockout and inhibitor studies, whereas in the resolution phase of disease, it plays a beneficial role.[93] Thus, MMP‐9 inhibition at the early inflammatory phase is desirable but inhibitors should have relatively short half‐lives so as to not hinder the beneficial effects of MMP‐9 during disease resolution.The THPI described herein includes 4 Gly‐Pro‐Hyp repeats on both the N‐ and C‐termini. Gly‐Pro‐Hyp repeats can served as binding elements for platelet glycoprotein VI (GPVI).[94] The (Gly‐Pro‐Hyp)2 repeat is the smallest motif that can be recognized by GPVI while the (Gly‐Pro‐Hyp)4 repeat is the smallest motif that activates GPVI.[95,96] This raises the issue of potential platelet activation by the THPI. Many of the prior GPVI studies utilize (Gly‐Pro‐Hyp)n sequences in multimeric presentation, as platelet activation has been reported as being dependent on collagen quaternary structure.[97] Crosslinked Gly‐Cys‐Hyp‐(Gly‐Pro‐Hyp)10‐Gly‐Cys‐Hyp‐Gly‐NH2 induced platelet adhesion, aggregation, and activation[95,96,98–102] but seemed to function in platelet signaling, rather than adhesion, under flow conditions.[103,104] Since the THPI is monomeric it would seem less likely to induce platelet activation then the crosslinked (Gly‐Pro‐Hyp)n sequences. However, Gly‐Cys‐Hyp‐(Gly‐Pro‐Hyp)10‐Gly‐Cys‐Hyp‐Gly‐NH2 in monomeric form can support adhesion and inhibit aggregation by collagen and is a partial GPVI agonist.[101,105] Thus, the THPI could induce some platelet activity, although it should be noted that the sequence surrounding Gly‐Pro‐Hyp can influence platelet interactions.[106] Mouse platelets are less active towards (Gly‐Pro‐Hyp)n sequences than human platelets[106] and thus the THPI utilized in the EAE model probably had little impact on platelet activity. Nonetheless, if THPIs are eventually to be considered for human applications additional studies are needed to determine their platelet reactivities.CONCLUSIONSThe present study has evaluated the effects of the α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI on T‐cell response to (a) general stimulation via CD3/CD28 and (b) specific antigen‐specific activation via the MOG35−55 peptide in an EAE model of MS. Immune cell proliferation and production of specific cytokines were modulated by the THPI. The previously determined specificity of the THPI indicates that its action is most likely via inhibition of MMP‐2 and/or MMP‐9. MMP‐9 activity is not only relevant for EAE and MS, but is related to the progression of many (neuro)inflammatory diseases.[107] The THPI presented herein may have much broader applications when applied at specific sites and within the appropriate window of opportunity.[107]ACKNOWLEDGMENTSThis work was supported by the Multiple Sclerosis National Research Institute (G.B.F.), a Neuroscience Pilot Award from the FAU Brain Institute, and the FAU Center for Molecular Biology & Biotechnology (CMBB). We thank Ms. Shannon Ortiz‐Umpierre for her assistance with the EAE studies and Drs. Manishabrata Bhowmick and Dorota Tokmina‐Roszyk for the synthesis of the THPI. The authors also acknowledge the Laboratory Animal Core at FAU and thank all the husbandry and veterinary staff for the care of our animal subjects of this study.CONFLICT OF INTERESTThe authors have no conflicts of interest to declare.DATA AVAILABILITY STATEMENTThe data that support the findings of this study are available from the corresponding author upon reasonable request.REFERENCESV. W. Yong, R. K. Zabad, S. Agrawal, A. G. DaSilva, L. M. Metz, J. Neurol. Sci. 2007, 259, 79.R. G. Rempe, A. M. Hartz, B. Bauer, J. Cereb. Blood Flow Metab. 2016, 36, 1481.T. Latronico, G. M. Liuzzi, Metalloproteinases Med. 2017, 4, 1.L. Muri, D. Leppert, D. Grandgirard, S. L. Leib, Cell. Mol. Life Sci. 2019, 76, 3097.A. Trivedi, L. J. Noble‐Haeusslein, J. M. Levine, A. D. Santucci, T. M. Reeves, L. L. Phillips, Cell. Mol. Life Sci. 2019, 76, 3141.D. Graesser, S. Mahooti, J. A. Madri, J. Neuroimmunol. 2000, 109, 121.H. Gerwien, S. Hermann, X. Zhang, E. Korpos, J. Song, K. Kopka, A. Faust, C. 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Application of a triple‐helical peptide inhibitor of MMP‐2/MMP‐9 to examine T‐cell activation in experimental autoimmune encephalomyelitis

Peptide Science , Volume 114 (5) – Sep 1, 2022

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Abstract

INTRODUCTIONThe active stage of multiple sclerosis (MS) is characterized by the breakdown of the blood–brain barrier (BBB), perivascular infiltration of inflammatory auto‐reactive T‐cells in the central nervous system (CNS), and demyelination. Matrix metalloproteinases (MMPs) are responsible for turnover and remodeling of the extracellular matrix (ECM) and CNS. The normal CNS contains low levels of MMPs, but several MMPs are upregulated in neurological disorders of the CNS, including MS.[1] MMPs are assumed to act as effector molecules in the (a) disruption of the BBB, including targeting of tight junction proteins (occludin, ZO‐1, and claudin‐5), basal lamina proteins (fibronectin, laminin, collagen, and heparan sulfate), chemokines, and growth factors,[2–5] (b) invasion of inflammatory cells into the CNS parenchyma,[2,3,6,7] (c) degradation of myelin‐associated glycoprotein (MAG) and myelin basic protein (MBP),[3,8] and (d) toxicity to axons and neurons.[1,9,10] MAG and MBP fragments are neuroantigens associated with the sensitization/activation of immune cells such as T‐cells. Sensitized T‐cells in the periphery are believed to cross the BBB and initiate a second wave of immune responses that activate microglia cells/macrophages and astrocytes to release proinflammatory cytokines and MMPs, contributing to further myelin degradation and axonal loss.[3,10] Imaging of MMP activity using a positron emission tomography‐compatible MMP inhibitor defined both relapsing and progressive active MS.[7]The application of synthetic MMP inhibitors in experimental autoimmune encephalomyelitis (EAE), a widely accepted animal model of MS, has demonstrated reduced clinical severity following administration after disease induction or onset of disease symptoms.[11–14] Effects were apparently mediated through restoration of the damaged BBB in the inflammatory stage of the disease[11] or reduction of leukocyte entry into the CNS,[12] although a lack of demyelination was observed in some cases.[13] A deficiency of tissue inhibitor of metalloproteinase (TIMP)‐1, an endogenous inhibitor of MMPs, in EAE mice results in persistent macrophage activation, myelin disruption, and increased disease severity.[10]Numerous MMPs have been implicated in MS. Of particular interest is the observation that MMP‐9 levels are elevated in MS patient serum, plasma, and cerebrospinal fluid, particularly in patients with active disease.[3,7,9,15–19] In addition, an increase in the MMP‐2:TIMP‐2 ratio marks chronic progression in MS while a high MMP‐9:TIMP‐1 ratio characterizes relapsing remitting MS.[1] Numerous studies have correlated increased MMP‐9 activity with the breakdown of the BBB, possibly through the degradation of tight junction proteins.[20–27] MMP‐9 derived from immune cells is critical for leukocyte penetration into the CNS parenchyma.[7] MMP‐9 inhibition reduces significantly the ability of naïve T‐cells to respond to stimulation.[28] MMP‐2, MMP‐9, MMP‐10, and MT6‐MMP cleave MBP efficiently.[29] Loss of both MMP‐2 and MMP‐9 results in the inability to induce EAE in mice,[30] while loss of MMP‐9 alone leaves young (but not older) mice significantly less susceptible to EAE initiation.[1,31] Hydrolysis of β‐dystroglycan by MMP‐2 and MMP‐9 promotes leukocyte penetration of the parenchymal basement membrane during EAE.[30] In a related study, a 7 day treatment of MMP‐2/MMP‐9/MMP‐14 inhibitor SB‐3CT[32,33] in a mouse model of ischemia was found to be neuroprotective.[34,35]The use of MMP inhibitors for MS treatment has precedent. Doxycycline and minocycline, which are broad‐spectrum MMP inhibitors with IC50 values in the range of 2–50 and 100–300 μM, respectively,[36] have been utilized in clinical trials of MS patients. For example, treatment of 16 relapsing–remitting MS patients with a doxycycline/interferon combination for 4 months (ClinicalTrials.gov Identifier NCT00246324) reduced brain lesions and MMP‐9 serum levels, improved Expanded Disability Status Scale values, and was considered safe and well‐tolerated.[2,37] In similar fashion, minocycline treatment of relapsing–remitting MS patients resulted in no new active lesions after 1 month with detection up to 6 months for 10 patients[38] while combination treatment of minocycline with glatiramer acetate in 44 patients showed reduced number of lesions by 63%–65% and lowered risk of relapse after 8–9 months compared with glatiramer treatment alone.[39] Additional clinical trials using minocycline for MS treatment are reportedly upcoming.[4] Future correlation of MMP activity with MS subtypes[40] may provide insight into patient‐specific treatment strategies.Our laboratory has developed selective, high affinity MMP inhibitors modeled on the triple‐helical structure of collagen.[14,41–46] These triple‐helical peptide inhibitors (THPIs) utilized phosphinate analogs of Gly‐Leu, Gly‐Ile, and Gly‐Val to bind to the MMP active site Zn(II).[41,42,44] α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI was selective for MMP‐2 and MMP‐9, with Ki values of 190 and 91 nM, respectively, at 25°C, and 2 and 1 nM, respectively, at 37°C.[14] When administered intraperitoneally at a dose of 12.43 μg/mouse per day starting from day 7, THPI treatment reduced the clinical severity of EAE from day 12 on.[14] The THPI treated EAE mice on average had less weight loss per day and increased their weight before the control group.[14] However, the mechanism by which the THPI improved clinical outcomes in EAE mice was unknown. In the present study we used the α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI to assess its efficacy in mitigating key immune cell mediators of MS, such as CD4+ T‐cells, by measuring cytokine secretion following THPI treatment. T‐cells, and specifically CD4+ T‐cells from relapsing–remitting MS patients, have been shown to produce MMP‐9 and utilize it to promote leukocyte migration into the CNS.[7,13,28,47–49] Anti‐CD3 stimulation of CD4+ T‐cells induced MMP‐9 expression and increased protein production and release.[28] Herein we assessed the in vitro impact of the THPI on the responses associated with CD4+ T‐cell activation, such as cellular proliferation and the secretion of the key cytokines, such as interleukin (IL)‐2, tumor necrosis factor α (TNF‐α), and interferon γ (IFN‐γ). We then applied the THPI in vivo in the EAE model to evaluate pre‐clinical response and splenocyte proliferation and production of cytokines.MATERIALS AND METHODSPeptide synthesis and purificationThe α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI [sequence (Gly‐Pro‐Hyp)4‐Gly‐mep‐Flp‐Gly‐Pro‐Pro‐GlyΨ{PO2H‐CH2}Val‐Val‐Gly‐Glu‐Gln‐Gly‐Glu‐Gln‐Gly‐Pro‐Pro‐Gly‐mep‐Flp‐(Gly‐Pro‐Hyp)4‐NH2, where Hyp = 4‐hydroxy‐L‐proline, mep = (2S,4R)‐4‐methylproline, and Flp = (2S,4R)‐4‐fluoroproline] was synthesized and characterized as previously described.[14]In vitro cell cultureCD4+ T‐cells were purified from spleen or lymph nodes of C57/Bl6 naïve mice via positive selection (CD4+ T‐cell isolation kit, Miltenyi Biotech) according to manufacturer's instructions. Purity was confirmed to be greater than 95%. CD4+ T‐cells were then cultured at a density of 1 × 106/mL in cRPMI, consisting of Roswell Park Memorial Institute (RPMI) 1640 plus 10% fetal bovine serum and 100 u/mL Pen‐Strep. To assess the impact of the THPI on CD4+ T‐cells, T‐cells were cultured for 24 h in the presence or absence of THPI followed by stimulation of the TCR using CD3/CD28 mAb Dynabeads (Invitrogen) as per manufacturer's instructions. In the case of splenocytes, the same culture conditions were utilized using a single cell suspension of the splenocytes from EAE or control mice. The splenocytes were treated in vitro with 10 μM THPI for 24 h, rinsed twice with RPMI, and stimulated with 50 μg/mL of MOG35−55 peptide for 96 h.Enzyme‐linked immunosorbent assayCell‐free culture supernatant from PBS or inhibitor treated CD4+ T‐cells or splenocytes were collected at 96 h and used for protein analysis of IL‐2, TNF‐α, IFN‐γ, IL‐6, and IL‐10 via enzyme‐linked immunosorbent assay (ELISA; all kits from BD Biosciences). The ELISA assays utilized mouse mAbs against recombinant IL‐2, IL‐6, IL‐10, and IFN‐γ and a purified mouse Ab against recombinant TNF‐α. Absorbance at λ = 450 nm with a wavelength correction at λ = 570 nm was measured with an H1 Biotek plate reader. The optical density values of samples recorded was converted to picogram using a standard curve obtained using known quantities of recombinant cytokines according to manufacturer's instructions.Toxicity assayCell toxicity was evaluated using the CellTox Green Cytotoxicity Assay (Promega) according to manufacturer's instructions. Fluorescence was recorded at λexcitation = 490 nm and λemission = 520 nm using an H1 Biotek plate reader.Proliferation assayProliferation was assessed using the CellTiter 96 AQueous proliferation assay (Promega) according to manufacturer's instructions. Absorbance at λ = 490 nm was recorded using an H1 Biotek plate reader.Murine model of MSAll procedures involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (protocol # TPI 12‐11 FL and # A18‐04, IBC # 2018‐269). Complete Freund's Adjuvant was produced by emulsifying Incomplete Freund's Adjuvant (Thermo Scientific) and M. tuberculosis H37Ra (Becton Dickinson, Franklin Lakes, NJ). MOG35−55 peptide (Met‐Glu‐Val‐Gly‐Trp‐Tyr‐Arg‐Ser‐Pro‐Phe‐Ser‐Arg‐Val‐Val‐His‐Leu‐Tyr‐Arg‐Asn‐Gly‐Lys‐OH) was synthesized using standard Fmoc solid‐phase chemistry[50] and RP‐HPLC purified to >90%. EAE was induced in female C57BL/6J mice (Jackson Laboratory; 10–12 weeks of age) by subcutaneously inoculating an emulsification of Complete Freund's Adjuvant and the MOG35−55 peptide (200 μg/mouse) on Day 0. Pertussis Toxin (Hooke's Laboratory, 320 ng) was introduced via intra‐peritoneal injection 3 and 24 h later. The control group received PBS/animal/day, while the treated group received 0.03 μg α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI/animal/day from day 7 by intra‐peritoneal injection. Disease severity (scored based on the Hooke's Laboratory scoring guide [www.hookeslab.com/protocols/eaeAI_C57BL6.html]) and weight change were recorded from Day 5 until sacrifice.RESULTSIn MS and its animal model, EAE, T‐cells become sensitized to neuroantigens by the concurrent stimulation of the T‐cell receptor (TCR) and its costimulatory molecule, CD28, in the presence of the neuroantigen. In vitro this process is effectively reproduced by the administration of CD3/CD28 antibodies.[28,51] Thus, to examine the effect of the THPI in T‐cell functionality, CD4+ T‐cells were enriched from the splenocytes of C57/BL6 mice, treated in the presence or absence of THPI, and stimulated with anti‐CD3/CD28 mAbs.THPI effects on proliferation and cytokine production of naïve T‐cellsOnce CD4+ T‐cells experience antigenic stimulation, subsets of these lymphocytes respond by proliferating and producing pro‐proliferation cytokines such as IL‐2 as well as the effector cytokines IFN‐γ and TNF‐α. Proliferation is thus a hallmark of a robust stimulation that helps to magnify the immune response and is aided by the autocrine actions of IL‐2. Thus, we sought to determine what impact THPI treatment would have on CD4+ T‐cell stimulation. THPIs need approximately 24 h incubation to adequately assume triple‐helical structure[52]; and we allowed an additional 24 h time frame once added to cells to allow for the reformation of the triple‐helical structure in the advent of any potential disruption associated with the cell culture process. CD4+ T‐cell toxicity due to the THPI was assessed and not observed (data not shown). A reduction trend in CD4+ T‐cell proliferation was observed at [THPI] = 25 μM (Figure 1).1FIGUREPurified CD4+ T‐cells were magnetically separated from the splenocytes of C57/BL6 naïve mice and pooled (n = 5) due to limited availability of samples. CD4+ T‐cells were then cultured with the THPI for 24 h, rinsed twice with RPMI‐1640, and then stimulated with CD3/CD28 mAbs in cRPMI for 72 h. Data is representative of two independent experimentsBased on the proliferation results, the impact of the THPI on cytokine production associated with T‐cell activation was determined after a 24 h incubation in culture and at a THPI concentration of 15 μM. IL‐2, which promotes CD4+ T‐cell proliferation upon CD3/CD28 activation,[53] was initially examined. There was a marked reduction in IL‐2 protein production (Figure 2 upper left panel) upon THPI treatment. The results indicated that the THPI hampered the ability of naïve CD4+ T‐cells to proliferate in response to antigenic stimulation.2FIGUREPurified CD4+ T‐cells were magnetically separated from the splenocytes of C57/BL6 naïve mice and pooled (n = 5) due to limited availability of samples. CD4+ T‐cells were then cultured with THPI for 24 h, rinsed twice with RPMI‐1640, and then stimulated with CD3/CD28 mAbs in cRPMI for 72 h. Cell free supernatant was collected and used to determine protein levels of IL‐2 (upper left), IFN‐γ (upper right) and TNF‐α (bottom) by ELISA. Data is representative of three independent experiments. T‐cells cultured with THPI for 24 h prior to 72 h CD3/CD28 stimulation significantly reduced production of IL‐2 (****p < 0.0001), IFN‐γ (****p < 0.0001), and TNF‐α (***p < 0.001). Statistical analysis was performed using a Student's t‐testTHPI treatment was next evaluated for effects on CD4+ T‐cell ability to produce effector cytokines that activate and/or stimulate other immune cells. In this case, the production of cytokines IFN‐γ and TNF‐α was quantified. IFN‐γ is an activator for the T‐cell effector phase and may directly induce apoptosis[54,55] while TNF‐α enhances CD4+ T‐cell proliferation and increases cytokine production.[56] THPI treatment of CD4+ T‐cells prior to CD3/CD28 stimulation reduced IFN‐γ and TNF‐α protein levels (Figure 2 upper right and bottom panels). These results suggested that the THPI can suppress CD4+ T‐cell effector function by reducing the production and/or secretion of IFN‐γ and TNF‐α.THPI treatment of EAE miceEAE is a well‐established murine model of MS, where the most widely used EAE model is the chronic progressive model in which C57BL/6J mice are immunized with myelin oligodendrocyte glycoprotein (MOG)‐derived peptide fragments.[57,58] EAE was induced herein with the MOG35−55 peptide.[13] Days 7–10 approximate the time of T‐cell activation and migration across the BBB which manifests as disease onset. Approximately 3–5 days after initial onset maximum clinical severity is observed.In contrast with our prior study,[14] we presently utilized the THPI at a dose (0.03 μg) more representative of practical application. Clinical symptoms of EAE appeared at day 10 and continued until the end of the experiment (day 20) (Figure 3, left panel). Untreated animals also exhibited weight loss (Figure 3, right panel). THPI treatment reduced the clinical severity of EAE from day 12 on (Figure 3, left panel). The THPI treated mice on average had less weight loss per day and increased their weight before the control group (Figure 3, right panel). Increased clinical severity and weight loss are hallmarks of EAE.3FIGUREResults of 0.03 μg α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI/day treatment of EAE mice. (left) Clinical score of THPI treated (gray) and nontreated (black) mice. (right) Weight compared with starting weight of THPI treated (gray) and nontreated (black) mice. n = 5 per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was performed using a two‐tailed Student's t‐testSplenocytes were isolated from THPI‐treated and untreated EAE mice at day 21. Splenocytes from THPI‐treated EAE mice were found to have reduced proliferation compared with untreated EAE mice (Figure 4, left panel). To determine if the reduced proliferation was due to cell death, isolated splenocytes were evaluated for toxicity. There was no significant difference in the degree of cytotoxicity between THPI‐treated and untreated EAE mice (Figure 4, right panel).4FIGUREAnalysis of splenocyte (left) proliferation and (right) cytotoxicity on day 21 following 0.03 μg α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI/day treatment of EAE mice. In vivo THPI administration reduced splenocyte proliferation in response to antigen, but the THPI was not toxic to splenocytes, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was performed by using a Student's t‐testTHPI effects on proliferation and cytokine production of splenocytes isolated from EAE miceT‐cells from diseased mice are known to have a different baseline cellular activity as compared with T‐cells from naïve mice who have not experienced any pathological conditions. We next assessed if THPI treatment would have similar effects on not just T‐cells from diseased mice, but on the overall immune response of which CD4+ T‐cells play a key role in orchestrating. Thus, splenocytes from EAE mice were isolated for an in vitro recall experiment.Following isolation, the splenocytes were treated with 10 μM of THPI for 24 h and either stimulated with 50 μg/mL of MOG35−55 peptide or not stimulated for 96 h. In addition to IL‐2 the cytokines IL‐10 and IL‐6 were monitored. IL‐10 is an anti‐inflammatory cytokine[59] while IL‐6 enhances CD4+ T‐cell proliferation and differentiates CD4+ T‐cells into subsets (see Section 4).[60,61] Production of IL‐2, IL‐10, and IL‐6 after 36 h was quantified. The unstimulated splenocytes had little (first two columns of Figure 5 upper left and upper right panels) to no (first two columns of Figure 5 bottom panel) cytokine production. MOG stimulated splenocytes (the third columns of Figure 5) produced significant amounts of IL‐2, IL‐10, and IL‐6. In turn, THPI treatment significantly reduced production of IL‐2 (fourth column of Figure 4 upper left panel), IL‐10 (fourth column of Figure 5 upper right panel), and IL‐6 (fourth column of Figure 5 bottom panel).5FIGURESplenocytes isolated from untreated EAE animals were treated with and without THPI for 24 h and stimulated with and without MOG33−35 for 96 h. Cell free supernatant was used in an ELISA to determine protein levels. The production of (top left) IL‐2, (top right) IL‐10, and (bottom) IL‐6 was significantly reduced in stimulated cells, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Statistical analysis was performed using a Student's t‐testSplenocytes were treated as described above and effector cytokine levels quantified. The unstimulated splenocytes did not produce significant amounts of IFN‐γ (first two columns of Figure 6 left panel); on the other hand, TNF‐α production from unstimulated splenocytes was evident and was reduced by THPI treatment (first two columns of Figure 6 right panel). The MOG stimulated splenocytes produced IFN‐γ (third column of Figure 6 left panel) and increased levels of TNF‐α compared with unstimulated splenocytes (first and third columns of Figure 6 right panel). IFN‐γ and TNF‐α levels were significantly reduced following THPI treatment (fourth column of Figure 6 both panels). The results indicated that THPI treatment could reduce the effector response of immune cells during EAE, thus mitigating disease severity.6FIGURESplenocytes isolated from untreated EAE animals were treated with and without THPI for 24 h and stimulated with and without MOG33−35 for 96 h. Cell free supernatant was used in an ELISA to determine (left) IFN‐γ and (right) TNF‐α protein levels. The production of IFN‐γ and TNF‐α was reduced, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was performed using a Student's t‐testDISCUSSIONThe present study was designed to initially evaluate if MMP‐2/MMP‐9 activity of T‐cells potentially contributed to EAE pathology. As a first step, a homogenous population of murine CD4+ T‐cells obtained from the spleen of naïve mice was pre‐treated with an MMP‐2/MMP‐9‐targeting THPI, followed by stimulation of the TCR using CD3/CD28 antibodies. A modest suppression of T‐cell proliferation at [THPI] = 25 μM was observed without cellular toxicity. We allowed for a 24 h time frame for the triple‐helix to form in the dilutions, and then added them directly to cells. The THPI concentration of 10–15 μM assured a well‐formed triple‐helix and the potential for an initial effect on CD4+ T‐cell proliferation.For the EAE studies the THPI dose used, in theory, approximates the α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI Ki for MMP‐2 and MMP‐9[14] based on the following calculation. With a molecular weight of 12.4 kDa, one 0.03 μg dosage of α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI corresponds to 2.4 × 10−12 mol. Mice have ~58.5 mL of blood per kg of bodyweight. Hence, a mouse weighing 25 g would have a total blood volume of ~58.5 mL/kg × 0.025 kg = 1.46 mL. Consequently, the blood concentration of α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI would reach 2.4 × 10−12 mol/1.46 × 10−3 L = 1.64 nM.THPI treatment of CD3/CD28 stimulated CD4+ T‐cells resulted in the reduction of the (a) initial proliferation of CD4+ T‐cells, (b) production of the proliferative cytokine IL‐2, and (c) production of effector cytokines IFN‐γ and TNF‐α. These indicators of T‐cell activity were subsequently utilized to evaluate the activity of the THPI in the EAE model of MS. The THPI reduced the clinical severity of EAE and splenocytes isolated from THPI‐treated EAE mice had reduced proliferation. In this latter case proliferation (Figure 4, left panel) is due to antigen‐specific (MOG35−55 peptide) activation, whereas in the former case proliferation (Figure 1) is due to general T‐cell stimulation. The two cases represent immunologically distinct circumstances which accounts for the modest effect observed with THPI inhibition of general T‐cell stimulation and the significant effect observed with THPI inhibition of antigen‐specific splenocyte activation.To determine if the THPI effects on CD4+ T‐cells was recapitulated in EAE mice, splenocytes were isolated, stimulated with auto‐antigen (MOG35−55), and treated with the THPI. Splenocytes capture a significant range of immune cells, 19%–22% CD4+ T‐cells, ~18% CD8+ T‐cells, ~2% CD4+ regulatory T‐cells, 18%–21% B cells, ~3% natural killer cells, ~2% immature myeloid cells, 2%–4% myeloid dendritic cells, 4%–5% macrophages, and 2%–3% neutrophils.[62] Similar effects were observed with splenocytes isolated from EAE mice compared with CD4+ T‐cells isolated from naïve mice. Of the cytokines examined, IL‐2 allows for a robust immune response via T‐cell proliferation, IL‐10 can downwardly regulate the immune response, and IL‐6 can shape the immune response in EAE to a highly aggressive form. As the immune response moves into the effector phase, cytokines such as IFN‐γ and TNF‐α become important for the destruction of target cells and recruitment of other immune cells. These data also suggest that other immune cells present in the splenocytes are sensitive to the effects of the THPI.The innate arm of the immune system (macrophages, dendritic cells, neutrophils) is typically the first to encounter an immunogen. In MS these cells phagocytize neuroantigens such as MBP and MOG and break them down into small fragments, for presentation to the TCR. This process sensitizes the T‐cells to the neuroantigen and establishes neuronal cells as target cells. In MS and EAE, T‐helper 1 (Th1) and Th17 cell subsets are the key mediators of pathological conditions.[63] MMP‐9 generates immunogenic epitopes that induce Th1 and Th17 cell subsets.[64]Naïve T‐cells and activated Th1 and Th2 cells produce cytokines such as IL‐2, IL‐6, IL‐10, and IL‐12, associated with the sensitization phase of EAE,[65–69] while activated Th1 and Th17 cells produce cytokines such as IL‐17, IFN‐γ, and TNF‐α, associated with the effector phase of EAE.[57] These cytokines influence the magnitude of the immune response in EAE and MS.[70,71] For example, TNF‐α is associated with successful stimulation of the TCR pathway and key mediation of immune cell assault on the CNS during EAE and MS. Thus, one way in which the THPI mitigated EAE clinical outcomes is by suppression of TNF‐α production (Figure 6).In the present study the treatment of activated CD4+ T‐cells with the MMP‐2/MMP‐9 selective THPI resulted in decreased cell proliferation and production of IL‐2, IFN‐γ, and TNF‐α (Figures 1 and 2). These results are consistent with prior reports. Treatment of T‐cells with minocycline, a broad‐spectrum MMP inhibitor (see below), resulted in decreased cell proliferation and production of IL‐2, IFN‐γ, and TNF‐α.[72] Treatment of anti‐CD3‐stimulated CD4+ T‐cells with MMP‐2/MMP‐9/MMP‐14 inhibitor SB3CT resulted in decreased cell proliferation, IL‐2 production, and CD25 expression.[28] Anti‐CD3‐stimulated CD4+ T‐cells isolated from MMP‐9 deficient mice (MMP9−/−) showed an 80%–85% decrease in proliferation, reduced IL‐2 production, increased intracellular calcium flux, and reduced CD25 and CD69 expression compared with CD4+ T‐cells from wild‐type mice.[28] In addition, anti‐CD3‐stimulated MMP9−/− CD4+ T‐cells showed increased CD62L, CTLA‐4, and CD45RO expression compared with CD4+ T‐cells from wild‐type mice.[28]A recent study examined the effects of naïve CD4+ T‐cell stimulation by CD3 and CD28 antibodies in the presence of collagen‐derived peptides Pro‐Hyp and Hyp‐Gly.[73] These peptides could presumably result from the action of MMP‐9, amongst other proteases, on collagen. Collagen peptide ingestion resulted in increased production of IL‐10 and IFN‐γ by splenocytes, but not IL‐2, IL‐6, or TNF, suggesting a shift towards a Th1‐dominant state.[73] The action of the peptides was through enhancement of STAT1 signaling. The effects observed in our study are different, in that the production of both IFN‐γ and TNF was suppressed by THPI treatment, and thus indicating that the presumed MMP‐2/MMP‐9 action herein was not through the same pathway as the degradation of ECM molecules.Although not identified here, one can consider the possible MMP‐2/MMP‐9 substrate(s) that result in modulation of T‐cell behaviors. The prior study on inhibition of T‐cell proliferation by SB3CT did not identify the MMP substrate but suggested that it was intracellular.[28] Utilizing a semi‐selective THPI with high affinity for MMP‐9, MMP‐9 was found to cleave citrate synthase in vitro and in vivo, generating a ~20 kDa fragment.[74] 5′‐adenosine monophosphate‐activated protein kinase α subunit has been identified as an MMP‐9 substrate in myeloid cells.[75] Both citrate synthase and 5′‐adenosine monophosphate‐activated protein kinase are key players in cellular energy homeostasis. Prior studies have identified numerous potential intracellular substrates for MMP‐2 and MMP‐9, including enzymes involved in carbohydrate metabolism and protein biosynthesis.[76] Alternatively, MMP‐2/MMP‐9 could be acting on T‐cells via processing cell surface biomolecules. It has been proposed that the major role of MMPs is modulating inflammatory and immune processes via processing of signaling molecules.[4,19,77] Along these lines, activation of T‐cells and subsequent production of IFN‐γ and IL‐2 was previously shown to be modulated by ADAM10 or ADAM17 cleavage of lymphocyte activation gene‐3 (CD223) from the cell surface.[78] We have applied machine learning in combination with RNA sequencing to uncover potential MMP‐2/MMP‐9 substrates in T‐cells[79] and will pursue this avenue to identify relevant MMP‐2/MMP‐9 substrates in EAE. The fact that all cytokines tested in the present study were reduced suggests that the MMP‐2/MMP‐9 substrate processing inhibited by the THPI impacts the cellular machinery in broad manner. A number of cytokines are known substrates of MMP‐2 and/or MMP‐9, such as interferon‐β (used for the treatment of MS), IL‐2, and monocyte chemoattractant protein‐3, and are functionally altered by the action of these proteinases.[80–82] Thus, of particular relevance here, in addition to indirect effects on adaptive immune processes, the inhibition of MMP‐9 can have a direct effect on the bioavailability of IL‐2.[82]As previously discussed,[14,83] THPIs have several favorable properties that facilitate their use as probes of enzyme activity and imaging agents in vivo, including the excellent stability of triple‐helical peptides in vivo[84–86] and the efficacy of MMP inhibiting phosphinic peptides in vivo.[87] The α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI structure facilitates binding of the inhibitor to the targeted MMPs while minimizing proteolytic degradation. Although MMP‐9 selective, inhibitory antibodies and antibody fragments have been described,[83,88] their application for neurological disorders is limited based on the inefficiency of antibodies to cross an intact BBB although they may cross a compromised BBB during active disease. One goal of using MMP inhibitors is to rapidly restore a compromised BBB (by minimizing proteolysis of tight junction proteins such as claudin‐5 and occludin[89]). Restoration of the intact BBB should allow small molecules to more efficiently enter the brain and inhibit additional detrimental processes in the CNS. The effectiveness of the MMP‐2/MMP‐9 α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI in the EAE mouse model indicated that this inhibitor could cross the BBB. Importantly, the inhibitory activities of the first generation THPI [C6‐α1(V)GlyΨ{PO2H‐CH2}Val THPI] were similar for full‐length (82 kDa) MMP‐9 and TIMP‐1 resistant (65 kDa) MMP‐9[42] and the TIMP‐1 resistant MMP‐9 appears to be the predominant enzyme form contributing to MS.[90–92]At the onset of EAE, MMP‐9 has a disease‐promoting effect, as evidenced by knockout and inhibitor studies, whereas in the resolution phase of disease, it plays a beneficial role.[93] Thus, MMP‐9 inhibition at the early inflammatory phase is desirable but inhibitors should have relatively short half‐lives so as to not hinder the beneficial effects of MMP‐9 during disease resolution.The THPI described herein includes 4 Gly‐Pro‐Hyp repeats on both the N‐ and C‐termini. Gly‐Pro‐Hyp repeats can served as binding elements for platelet glycoprotein VI (GPVI).[94] The (Gly‐Pro‐Hyp)2 repeat is the smallest motif that can be recognized by GPVI while the (Gly‐Pro‐Hyp)4 repeat is the smallest motif that activates GPVI.[95,96] This raises the issue of potential platelet activation by the THPI. Many of the prior GPVI studies utilize (Gly‐Pro‐Hyp)n sequences in multimeric presentation, as platelet activation has been reported as being dependent on collagen quaternary structure.[97] Crosslinked Gly‐Cys‐Hyp‐(Gly‐Pro‐Hyp)10‐Gly‐Cys‐Hyp‐Gly‐NH2 induced platelet adhesion, aggregation, and activation[95,96,98–102] but seemed to function in platelet signaling, rather than adhesion, under flow conditions.[103,104] Since the THPI is monomeric it would seem less likely to induce platelet activation then the crosslinked (Gly‐Pro‐Hyp)n sequences. However, Gly‐Cys‐Hyp‐(Gly‐Pro‐Hyp)10‐Gly‐Cys‐Hyp‐Gly‐NH2 in monomeric form can support adhesion and inhibit aggregation by collagen and is a partial GPVI agonist.[101,105] Thus, the THPI could induce some platelet activity, although it should be noted that the sequence surrounding Gly‐Pro‐Hyp can influence platelet interactions.[106] Mouse platelets are less active towards (Gly‐Pro‐Hyp)n sequences than human platelets[106] and thus the THPI utilized in the EAE model probably had little impact on platelet activity. Nonetheless, if THPIs are eventually to be considered for human applications additional studies are needed to determine their platelet reactivities.CONCLUSIONSThe present study has evaluated the effects of the α1(V)GlyΨ{PO2H‐CH2}Val [mep14,32,Flp15,33] THPI on T‐cell response to (a) general stimulation via CD3/CD28 and (b) specific antigen‐specific activation via the MOG35−55 peptide in an EAE model of MS. Immune cell proliferation and production of specific cytokines were modulated by the THPI. The previously determined specificity of the THPI indicates that its action is most likely via inhibition of MMP‐2 and/or MMP‐9. MMP‐9 activity is not only relevant for EAE and MS, but is related to the progression of many (neuro)inflammatory diseases.[107] The THPI presented herein may have much broader applications when applied at specific sites and within the appropriate window of opportunity.[107]ACKNOWLEDGMENTSThis work was supported by the Multiple Sclerosis National Research Institute (G.B.F.), a Neuroscience Pilot Award from the FAU Brain Institute, and the FAU Center for Molecular Biology & Biotechnology (CMBB). We thank Ms. Shannon Ortiz‐Umpierre for her assistance with the EAE studies and Drs. Manishabrata Bhowmick and Dorota Tokmina‐Roszyk for the synthesis of the THPI. 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Journal

Peptide ScienceWiley

Published: Sep 1, 2022

Keywords: experimental autoimmune encephalomyelitis; matrix metalloproteinase; multiple sclerosis; T‐cell activation; triple‐helical peptide inhibitor

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