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Myxomavirus-derived serpin prolongs survival and reduces inflammation and hemorrhage in an unrelated lethal mouse viral infection - PubMed

Myxomavirus-derived serpin prolongs survival and reduces inflammation and hemorrhage in an unrelated lethal mouse viral infection

Hao Chen et al. Antimicrob Agents Chemother. 2013 Sep.

Abstract

Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c(+) splenocytes (macrophage and dendritic cells) and reduced CD11b(+) tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.

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Figures

Fig 1
Fig 1

Serp-1 prolongs survival in MHV68-infected IFN-γR−/− mice and in wild-type ebolavirus-infected BALB/c mice. (A) Treatment with Serp-1 (100 μg/kg/day) for 10 days beginning immediately after MHV68 infection in IFN-γR−/− mice prolonged early survival (P ≤ 0.048) by approximately 10 days (n = 60 mice). (B) Treatment with NSP given immediately after MHV68 infection for 10 days did not improve survival, with a trend toward increased mortality compared to saline (P = 0.079; n = 12). (C) Treatment with Serp-1 for the first 30 days postinfection, beginning on the day of infection, further prolonged survival in MHV68-infected IFN-γR−/− mice compared to the saline-treated mice (P ≤ 0.02; n = 10). (D) Treatment for 10 days with Serp-1 beginning 21 days after MHV68 infection produced only a nonsignificant trend toward improved survival (P = 0.326; n = 12). (E) Treatment with Serp-1 for 30 days beginning 7 days postinfection again significantly improved survival (P ≤ 0.025; n = 12). (F) Serp-1 treatment at 100 μg/kg/day similarly improved survival in ebolavirus-infected BALB/c mice but did not alter survival at lower doses (ANOVA, P ≤ 0.011) (Kaplan-Meier survival analysis).

Fig 2
Fig 2

Serp-1 treatment reduces detectable MHV68 antigen expression in IFN-γR−/− mice at 10 days' follow-up and ZEBOV GP expression (FFU equivalents) for ebolavirus in BALB/c mice at 14 days' follow-up. Immunohistochemical staining for MHV68 at 10 days' follow-up demonstrated positively stained mononuclear cells (brown) in aortic (A), lung (C), and colon (E) histologic sections from saline-treated IFN-γR−/− mice. Positively stained cells were detected predominantly in the aortic adventitial layer (A), in interstitial and bronchial epithelial layers in the lung (C), and in submucosa and serosa in the colon (E). Positively stained cells (mean count per high-power field) were significantly reduced with Serp-1 treatment in lung (D and G) and colon (F and G) sections (P ≤ 0.004 and P ≤ 0.015, respectively; n = 14), with only borderline reductions in aorta (B and G). Arrowheads indicate cells staining positively for MHV68. FFU equivalents for ebolavirus were also reduced with Serp-1 treatment at 100-μg/kg doses for 10 days but not with lower Serp-1 doses at 6 days' follow-up (H) (n = 31). SE cannot be calculated for the ebolavirus FFU measurements, as only 2 representative mice were tested. For MHV68-positive IHC-stained sections, the magnification is ×400. Insets represent higher-power (×1,000) magnification.

Fig 3
Fig 3

Large inflammatory cell infiltrates were found in the aortic adventitia (A) and lung (C) in saline control-treated MHV68-infected IFN-γR−/− mice at 10 days' follow-up. Serp-1 treatment significantly reduced cell counts in aortic sections (A, B, and J) (P ≤ 0.01; n = 14). Inflammatory cells infiltrates accompanied by hemorrhage and alveolar consolidation are seen in lung sections from saline-treated MHV68-infected control mice (C). Infiltrating cells were reduced after Serp-1 treatment (D and J) (P ≤ 0.047). Serp-1 treatment produced a nonsignificant reduction in inflammatory cells (F and J) (P = 0.27) in colon sections compared to saline (E). Inflammatory cell counts were increased in the aortic adventitia (G and K) (P ≤ 0.02), lung (H and K) (P ≤ 0.009), and colon (I and K) (P ≤ 0.018) in NSP-treated MHV68-infected mice compared to saline controls. Bar graphs demonstrate numbers of invading cells per high-power field, three fields counted per section in Serp-1-treated (J) and NSP-treated (K) MHV68-infected mice compared to saline controls (n = 14). Arrowheads indicate the mononuclear cell infiltrates. Thick arrows indicate lung hemorrhage. Cell count is indicated as mean per high-power field ± SE for H&E-stained sections. Magnification, ×400.

Fig 4
Fig 4

Serp-1 inhibits invasion of CD11b+ but not CD3+ cells into the aorta and lungs in MHV68-infected IFN-γR−/− mice at 10 days' follow-up (n = 14). Increased numbers of invading CD11b+ cells were found in the aortic adventitia (A), lung (B), and colon (C) in saline-treated control mice. Serp-1 treatment significantly reduced CD11b-positive cell counts in the aorta (D and G) (P ≤ 0048) and lung (E and G) (P ≤ 0005) but not the colon (F and G) (P = 0.098) compared to saline controls. CD3+ cells were detected in the aortic adventitia, lung, and colon in saline-treated mice, with smaller numbers in the lung and with nonsignificant decreases in Serp-1-treated mice (H) (P = 0.197, 0.172, and 0.49, respectively) (n = 14). Positively stained cells are indicated as mean count per HPF ± SE. Arrows indicate CD11b+ cells. Magnification, ×400. Insets represent higher-power (×1,000) magnification.

Fig 5
Fig 5

Serp-1 reduced spleen and liver lesions in mouse-adapted Zaire ebolavirus infections in BALB/c mice. H&E-stained sections of spleen (A and B) and liver (C and D) from ebolavirus-infected mice are shown. Control saline-treated sections demonstrate the severe lymphoid hyperplasia of the periarteriolar sheaths (PAL), particularly the marginal zone (arrows) in the spleen (A) that obscures much of the red pulp (R) in infected mice. More normal splenic architecture is seen in the Serp-1 (100 μg/kg/day)-treated mice (B). Moderate to severe necrosis and accompanying inflammation in the periportal and midzonal hepatocytes (arrows) with evidence for severe disruption of the lobular architecture and relative sparing of the central lobular hepatocytes in saline-treated mice can be seen. There is widespread microvesicular lipid degeneration (arrows) and hepatocellular necrosis (arrowheads) disrupting the hepatic cords in control-treated mice (C). Serp-1 markedly reduced evidence for necrosis and tissue damage in hepatic sections (D). P, portal; CV, central vein. Magnification, ×100. Bar graphs demonstrate a clear dose-dependent reduction in spleen lymphoid necrosis (E), marginal zone hyperplasia (F), vascular macrophage invasion (G), and hepatocyte degeneration (H) with Serp-1 treatment at the higher doses. Histology was scored by J. Abbott, blinded to treatment, using a scale of 0 to 5, with increasing severity at higher numbers (n = 31); data represent mean scores ± SEs. Magnification, ×100.

Fig 6
Fig 6

Serp-1 and NSP had opposing effects on splenocyte responses assessed by flow cytometry. Serp-1 significantly increased CD11c counts at 10 days postinfection (A) (P ≤ 0.045; n = 14). Conversely, NSP reduced CD11c (macrophages and DCs; P ≤ 0.010), CD4 IL-4 (Th2 cells; P ≤ 0.023), CD3 CD8 (Tc cells; P ≤ 0.002), CCR6 (nonactivated memory T cells and DCs; P ≤ 0.030), CD19 (B cells; P ≤ 0.008), CD83 (mature DCs; P ≤ 0.01), and CD206 (macrophages and DCs; P ≤ 0.035) cells at 10 days (B) (n = 14). Cell counts are presented as means ± SEs. *, **, and ***, P ≤ 0.05, P < 0.01, and P < 0.009, respectively.

Fig 7
Fig 7

Serp-1 and NSP yielded opposing changes on coagulation and inflammatory pathway gene expression and activity in MHV68-infected mouse aortas at 10 days (n = 14). Serp-1 increased gene expression for IL-10 (P ≤ 0.001), bFGF (P ≤ 0.022), addressin (P ≤ 0.001), PAI-1 (P ≤ 0.021), and NSP (P ≤ 0.009) and decreased IL-6 (P ≤ 0.016) and PAI-2 (A) (P ≤ 0.016), compared to NSP. On immunostained sections, Serp-1 (B) increased IL-10 levels (ELISA) in plasma samples from MHV68-infected mice (P ≤ 0.032), while NSP reduced IL-10 levels (F) (P ≤ 0.022). Bleeding time was increased in MHV68-infected mice compared to noninfected mice (C) (P ≤ 0.016, n = 14). Serp-1 and NSP both decreased bleeding time compared to that in MHV68-infected saline controls (P ≤ 0.046 and P ≤ 0.011, respectively; n = 21). Serp-1 increased bleeding time compared to NSP (P ≤ 0.017). Serp-1 reduced fXa staining in aortic sections compared to control MHV68-infected mice (E and F) at 10 days compared to saline controls (D and F) (P ≤ 0.016; n = 6). All values are provided as means ± SEs; arrows indicate fXa staining. Magnification, ×400. Insets represent higher-power (×1,000) magnification. *, **, and ***, P ≤ 0.05, P < 0.01, and P < 0.009, respectively.

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