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Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines in vitro - PubMed

Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines in vitro

Kasinath Viswanathan et al. PLoS One. 2012.

Abstract

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.

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Conflict of interest statement

Competing Interests: ARL is the chief clinical officer and holds shares in a small Biotechnology company, Viron Therapeutics, Inc in London, Canada, which is developing another viral serpin for clinical use. The cross-class serpins in this paper have different molecular targets and are not in any way connected to, nor under development by, this biotech company. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Serp-2 reduces plaque growth in arterial surgery models.

Treatment of rat models of arterial injury with viral cross-class serpins demonstrates reduced vasculopathy. Haematoxylin and eosin stained sections of rat iliofemoral arterial sections harvested at 4 weeks and the mean plaque area were measured and presented as mean ± SE. The results demonstrated reduced plaque growth with Serp-2 (6 rats/dose; 3–3000 ng total N = 24) treatment at doses >30 ng (A). CrmA (6–12 rats/dose, 0.03–3000 ng; total N = 60) (B) and the Serp-2 reactive center loop mutant D294E (6 rats/dose, 0.3–3000 ng; total N = 18) treatments (C) demonstrated a non-significant trend toward reduced plaque at 30 ng, with no significant inhibition of plaque growth at higher concentrations. (D) In the mouse aortic allograft transplant model (PAI-1−/−, B6.129S2-Serpine1tm1Mlg donor to Balb/C recipient) Serp-2 again significantly reduced plaque (p<0.026), whereas CrmA and the D294A and D294E Serp-2 RCL mutants did not reduce plaque (total mice 33). (E–H) Five micron thick cross sections of mouse aortic allograft transplants taken from within the transplanted donor aortic section, demonstrate the marked intimal hyperplasia and associated mononuclear cell invasion in the adventitial layers in saline (E) or CrmA (F) treated mice. Treatment with Serp-2 (G) but not Serp-2 D294E mutants (H), displayed reduced plaque and inflammation. Arrows bracket intimal plaque limits. Arrowheads point to areas of mononuclear cell invasion. Magnification 100X.

Figure 2
Figure 2. Serp-2 reduces plaque growth in Apolipoprotein E deficient (ApoE−/−) mice.

Hyperlipidemic ApoE−/− mice were infused with a bolus of Serp-2, CrmA or control saline after carotid cuff compression injury. Histological sections taken at the aortic root, where no surgical injury occurs, and distally at the site of cuff injury were examined. Cross sections taken at the aortic valve level (Oil red O staining) demonstrate plaque growth in saline treated control mice (A) (N = 11). A significant reduction in plaque area is detectable with Serp-2 (B, N = 11), but not with CrmA (C, N = 11) treatment compared to saline treated controls. Morphometric analysis of plaque area at the aortic root in ApoE−/− mice, where no surgery has been performed, demonstrated that Serp-2 inhibited aortic plaque and macrophage/foam cell invasion (D, p<0.001) to a greater extent than at sites of vascular carotid compression surgery in the same model (E, P = 0.06). Oil red O staining confirmed a reduction in fatty plaque in the ApoE−/− aortic root (F), indicating decreased foam cell/macrophage invasion (p<0.001). Thin arrows bracket intimal plaque limits, larger arrows identify an aortic leaflets, large arrow with open base points to area of fatty, foam cell (macrophage) invasion; Magnification – 100X.

Figure 3
Figure 3. Viral cross-class serpins alter apoptotic responses in T cells and monocytes, in vitro.

Apoptotic responses were induced in T cells and monocytes using camptothecin or staurosporine. Inhibition of caspase 3 and 7 activity was measured after treatment with Serp-2, CrmA, or the two Serp-2 mutants by analysis of changes in DEVDase activity, with comparison to untreated controls. Serp-2, but not CrmA nor D294A and D294E treatment of Jurkat T cells reduced caspase 3 activity (DEVDase) after camptothecin (CPT) (A, p≤0.001) or staurosporine (STS) (B, p≤0.033) apoptosis actuator treatment. Cell death in T cells measured as fragmented DNA by ELISA was also blocked in CPT treated T cells (C, p≤0.012). Serp-2 (p≤0.032) and CrmA (p≤0.001) both significantly reduced CPT-induced elevations in caspase 3 and 7 activity in monocytes (D). The results shown here represent mean ± SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.

Figure 4
Figure 4. Blockade of granzyme B reduces viral cross-class serpin inhibition of T cell induced T cell apoptosis.

Jurkat T cells were treated with PMA and ionophore (PI) and the level of granzyme B expressed was measured by IEPDase assay and caspase 3 and 7 activity by DEVDase assay (A). Increased granzyme B (GzmB, p<0.001) secreted by these cells and was inhibited by treating the cells with an intracellular inhibitor of granzyme B, ZAAD-CMK (p<0.001) or anti-granzyme B antibody (p<0.001) (A). The medium containing granzyme B from PI treated T cells (CTLm) was applied to naive HUVECs to induce apoptosis (B). Treatment with Serp-2, but not CrmA, reduced caspase activity in CTLm treated HUVECs (B). The CTLm was also applied to naive T cells in culture to induce apoptosis and increased levels of caspase 3 and granzyme B were observed as IEPDase activity (C, p<0.0001) and DEVDase activity (D, p<0.0001) respectively. Treatment with Serp-2 reduced both granzyme B (C, p<0.01) and caspase 3 (D, p<0.0009) activities significantly. Antibody to granzyme B (GzmB) blocked Serp-2 mediated reductions in CTLm induced granzyme B (C) when compared to Serp-2 treatment alone (p<0.0004), but with a still significant decrease (p<0.005) when compared to CTLm treatment alone. Antibody to granzyme B also blocked the Serp-2 mediated decrease in caspase 3 (D, p<0.149) when compared to CTLm activation. This Serp-2 mediated inhibition of CTLm induced granzyme B activity was also blocked by incubation of cells with antibody to perforin (C, p = 0.412) but not the caspase 3 activity (D, p<0.0193). The results shown here represent mean ± SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.

Figure 5
Figure 5. Serp-2 binds T cells in vitro with greater affinity than CrmA.

Jurkat T cells were treated with FITC labeled Serp-2 or CrmA and binding/ association of these viral proteins with T cells was analyzed using Flow cytometry (FACS) analysis (A) and fluorescence microscopy (B, Magnification 10X). Extracellular, surface FACS analysis shows both Serp-2 and CrmA bind to the T cell surface (A). This observation was also supported by the fluorescent microscopic analysis (B). Intracellular ZAAD-CMK granzyme B inhibitor decreased Serp-2 mediated inhibition of caspase 3 activity in response to treatment with CTLm (cytotoxic-like T cell medium) (C, p<0.005). Treatment with antibody to granzyme B (α GzmB) or perforin (α PF) partially blocked Serp-2 binding (D, p<0.003 and p<0.012, respectively). The results represent mean ± SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.

Figure 6
Figure 6. Granzyme B deficiency (GzmB−/−) in donor aorta interferes with Serp-2 inhibition of plaque growth after aortic allograft transplant.

ApoE−/− (C57Bl/6) donor aortic allograft transplant into Balb/C recipient mice (N = 16) induced plaque growth at 4

weeks follow up as measured by plaque area (A) and intimal to medial thickness (IMT) ratios (B). Serp-2 treatment significantly reduced plaque area (A, p<0.036) and IMT ratios (B, p<0.045) when compared to saline treatment. CrmA treatment markedly increased plaque area (A, p<0.026) and non-significantly increased IMT ratios (B, p = 0.312). Saline treated ApoE−/− GzmB−/− DKO allografts (N = 18) had non-significant reductions in plaque area and IMT when compared to ApoE−/− donor allografts (A, B). CrmA treated ApoE−/− GzmB−/− DKO allografts had significantly reduced plaque area (A, p<0.021) and IMT (B, p<0.006) when compared to CrmA treated ApoE−/− donor allografts. Serp-2 no longer reduced plaque area (A) or IMT (B) in either GzmB−/− allografts (p = 0.995 for plaque area and p = 0.992 for IMT) or ApoE−/− GzmB−/− DKO (p = 0.704 for plaque area and p = 0.353 for IMT). Serp-2 but not Serp-2 mutant D294E significantly reduced caspase 3 staining in all three arterial layers (Fig6C, p<0.024) in PAI-1−/− mice 4weeks post-aortic transplant. Immunostained sections for caspase 3 illustrate reduced staining in mononuclear cells in Serp-2 treated PAI-1−/− aortic transplants when compared toD294E treatment (Fig6D, Mag 400X).
Figure 7
Figure 7. CD3 and active Caspase 3 populations 72 hrs after mouse aortic allograft.

C57Bl/6 donor aortic allografts were transplanted into Balb/C recipient mice (N = 3 per treatment) and followed up at 72

hrs. Compared to saline, Serp-2 but not CrmA treatment reduced caspase 3 activity (panels A-C,G; p<0.0224). Neither protein treatment significantly reduced CD3+ T cells (panels D-F,H).

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