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We found that platelet depletion reduces intrahepatic accumulation of virus-specific cytotoxic T lymphocytes (CTLs) and organ damage in mouse models of acute viral hepatitis. Transfusion of normal but not activation-blocked platelets in platelet-depleted mice restored accumulation of CTLs and severity of disease. In contrast, anticoagulant treatment that prevented intrahepatic fibrin deposition without reducing platelet counts did not avert liver injury. Thus, activated platelets contribute to CTL-mediated liver immunopathology independently of procoagulant function.
Hepatic damage resulting from infection by nonlytic viruses, such as hepatitis B virus (HBV) and replication-deficient adenoviruses, is a consequence of the antigen-specific CTL response1,2 aimed at viral clearance. We show here that platelets, which participate with leukocytes in inflammatory reactions3,4, are involved in CTL-induced immunopathology and antiviral activity.
We initially observed that HBV transgenic mice injected with HBV-specific CTLs and C57BL/6J mice infected with a replication-deficient adenovirus expressing lacZ (RAd35) showed platelet aggregates in the liver alongside apoptotic hepatocytes and inflammatory cells (Supplementary Fig. 1 online). To verify whether platelets contribute to the development of these lesions, we injected mice with antibodies (α-PLT) against mouse glycoprotein (GP) Ibα (Supplementary Methods online) that caused a >97.5% reduction in platelet count within 30 min and for up to 6 d (Supplementary Table 1 online). Platelet counts remained normal (8–10 × 105/μl of blood) in mice that received saline (NaCl) solution or an irrelevant antibody (α-Irr). We then evaluated liver disease severity in Thy-1.2+ HBV transgenic mice injected with α-PLT or α-Irr before transfer of Thy-1.1+ HBV-specific CTLs. In agreement with previous results5–8, the liver of α-Irr–treated mice killed at the peak of serum alanine aminotransferase (sALT) elevation showed scattered necroinflammatory foci, in which loss of hepatocytes (hepatocellular dropout) was readily detectable (Fig. 1a). The degree of hepatocellular dropout as well as the size of necro-inflammatory foci (mean size, 1,245 μm2 versus 6,724 μm2; Fig. 1b) was smaller in thrombocytopenic mice, which also showed 80% lower sALT levels (Fig. 1c) and ~40% (P < 0.01) and 30% (P < 0.01) fewer intrahepatic Thy-1.1+ HBV-specific CTLs at days 1 and 2 after transfer, respectively (Fig. 1d). CTL function was unaffected by the low platelet count, as Thy-1.1+ HBV-specific CTLs recovered from the liver of normal or thrombocytopenic mice had identical cytotoxic activity against target cells that express hepatitis B surface antigen (Fig. 1e) and identical interferon (IFN)-γ (encoded by Ifng) production ex vivo (data not shown). In thrombocytopenic mice, intrahepatic Ifng mRNA (a marker of antigen recognition by CTL6,9) was reduced (Supplementary Fig. 2 online) in proportion to the number of CTLs in the liver (Fig. 1d), but probably yielded sufficient levels of this antiviral cytokine6,9 to abolish viral replication (Supplementary Fig. 2 online).
To investigate further the role of platelets in CTL-induced liver disease, we transfused thrombocytopenic mice with washed mouse platelets lacking endogenous but expressing human GP-Ibα (Supplementary Methods online). These platelets (TKK-PLT) are not recognized by α-PLT (Supplementary Fig. 3 online) and function normally in mice10. Transfusion of 6 × 108 TKK-PLTs into C57BL/6J mice treated with α-PLT 1 d earlier caused platelet counts of ~40% (at 0.5, 3 and 24 h) and ~22% (at 48 h) of NaCl-injected mice, whereas the same number of C57BL/6J-derived platelets yielded counts <3% of control as early as 0.5 h after transfusion (Supplementary Fig. 3 online). TKK-PLT transfusion restored severity of liver disease, intra-hepatic accumulation of CTL and levels of Ifng mRNA (Supplementary Fig. 4 online), unless TKK-PLTs were treated with the activation inhibitor prostaglandin (PG) E1 before transfusion (Fig. 1f and data not shown). Unlike untreated TKK-PLTs, circulating PGE1-treated TKK-PLTs isolated at autopsy did not aggregate in response to collagen stimulation in vitro (data not shown). Thus, activated platelets contribute to CTL-mediated immunopathological responses. Accordingly, we found that CTLs adhere to platelets activated on a model-reactive surface ex vivo (Supplementary Video 1 online).
Livers from HBV transgenic mice that received HBV-specific CTLs after injection of α-Irr showed abundant fibrin deposition as compared to NaCl-injected mice; fibrin deposits were reduced in mice rendered thrombocytopenic before CTL injection, but restored in thrombocytopenic mice that received TKK-PLT transfusion before CTL injection (Supplementary Fig. 5 online). This indicates that platelets are required for fibrin deposition. Treatment with the anticoagulant warfarin at a dose that caused a tenfold increase in whole-blood clotting time before CTL transfer also prevented liver fibrin deposition but had no impact on liver damage (Fig. 1g), CTL accumulation or intrahepatic Ifng mRNA levels (data not shown). Fibrin deposition, therefore, is a consequence and not a cause of CTL-induced liver injury.
To confirm our findings in a different model of acute viral hepatitis, we immunized C57BL/6J mice with a plasmid expressing lacZ before infecting them with RAd35. This allowed us to measure the pathogenic lacZ-specific CTL response and define the role of platelets independently of CTL priming. Mice with normal platelet counts killed at the peak of sALT elevation showed an overwhelming disease resembling fulminant hepatitis in humans (Fig. 2a). Thrombocyto-penia markedly improved this picture, with most hepatocytes retaining normal cytological appearance (Fig. 2b), paralleled by a ~90% reduction in sALT activity (Fig. 2c), and an 88% (P < 0.01) and 66% (P < 0.01) reduction in the intrahepatic accumulation of lacZ-specific CTLs at days 3 and 4 after infection, respectively (Fig. 2d). The ability of CTLs recovered from the liver of thrombocytopenic mice to express granzyme B or to produce IFN-γ ex vivo was not impaired (data not shown). Accordingly, intrahepatic Ifng mRNA levels in thrombocytopenic and control mice (Fig. 2e) paralleled the number of intrahepatic CTLs at the same time points (Fig. 2d). By day 5, thrombocytopenic mice showed a more abundant expression of lacZ RNA (Fig. 2f) and higher percentage of β-galactosidase–positive hepatocytes (Fig. 2g), an indication that RAd35 was not readily cleared. This suggests that, in the absence of platelets, the number of functionally normal lacZ-specific CTLs that had accumulated in the liver was not sufficient for viral clearance within the time frame of our experiments. As occurred in HBV transgenic mice, TKK-PLT transfusion in RAd35-infected thrombocytopenic mice restored severity of liver disease, intrahepatic accumulation of CTLs, levels of Ifng mRNA (Supplementary Fig. 4 online) and deposition of fibrin (Supplementary Fig. 5 online).
In conclusion, our findings show that platelet activation is necessary to accumulate virus-specific CTLs at the site of inflammation, thus mediating immunopathogenic responses. Events of this kind may occur during liver infections caused by noncytopathic viruses (such as hepatitis B and C viruses), in which CTLs have a crucial role in the progression of organ damage1,11,12. The experiments described in this report were approved by the Animal Research Committee of The Scripps Research Institute.
Supplementary Figure 1. Platelets accumulate within necroinflammatory foci of the liver. Immunohistochemical staining for mouse GP-Ibα (α-PLT) in the livers of HBV transgenic mice (lineage 1.3.32) that were killed 2 d after injection of NaCl (a) or HBV-specific CTLs (b); or from C57BL/6J mice that were killed 6 d after infection with RAd35 (c). Platelet aggregates (arrows) and cells displaying the histological features of apoptotic hepatocytes (arrowheads) are indicated. The insets show a magnification of areas of interest. (Immunoperoxidase stain for α-PLT; scale bars, 50 μm [panels] and 25 μm [insets]).
Supplementary Figure 2. Reduced levels of IFN-γ expression and inhibition of viral replication reflect the homing of fewer but functional CTLs in the liver of thrombocytopenic HBV transgenic mice. (a) Total hepatic RNA derived from the same mice described in the legend of Figure 1d was analyzed by RPA for the expression of IFN-γ. The housekeeping mRNA encoding the ribosomal protein L32 was used to normalize the amount loaded in each lane, and quantitative phosphor imaging analysis was performed. The indicated numbers (mean ± standard deviation of 5 mice per group) were obtained by dividing each Ifng mRNA value by the amount of the corresponding L32 RNA. (b) Total hepatic DNA isolated from five representative CTL-injected mice and three NaCl-injected controls, sacrificed at d 2 after injection, was analyzed for HBV replication by Southern blot (lower) and quantitative phosphor imaging (upper). The relative content of HBV replicative intermediates (including the relaxed-circular [RC] and single-stranded [SS] linear HBV DNA) is expressed as arbitrary units calculated after normalization to the band corresponding to the integrated transgene (Trans.).
Supplementary Figure 3. TKK-PLTs are not recognized by α-PLT both in vitro and in vivo. Platelets from C57BL/6J mice (C57BL/6J PLT) (a) or mGP-Ibαnull;hGP-IbαTg mice (TKK-PLT) (b) were analyzed by FACS analysis using anti-human GP-Ibα and α-PLT (anti-mouse GP-Ibα). (c) Two groups of C57BL/6J mice (n = 4) that received a single dose of α-PLT were injected 24 h later with either TKK-PLTs (6 × 108/mouse, open triangles) or C57BL/6J PLTs (6 × 108/mouse, solid squares). Blood samples were collected at the indicated time points after platelet reconstitution and platelets counts are expressed as mean ± standard deviation. Blood samples from C57BL/6J mice injected with NaCl (gray circles) served as controls.
Supplementary Figure 4. Restoration of liver disease severity, CTLs accumulation and intrahepatic IFN-γ expression in platelet-depleted mice upon TKK-PLT transfusion. (a-d) Groups of HBV transgenic mice (n = 4) were injected either with α-Irr (white bars) or α-PLT (black and red bars) 24 hrs before injection of HBV CTLs. One group (red bars) also received TKK-PLT 4 h later. Mice receiving NaCl (dark gray bars) or α-PLT and TKK-PLT but no CTLs (light gray bars) served as controls. All animals were killed simultaneously, 1 d after CTL injection. Serum ALT (a), absolute numbers of intrahepatic CTLs (b) and Ifng mRNA (c) are expressed as mean ± standard deviation. (d) The platelet counts (mean ± standard deviation) in blood samples collected from all the animals in each group at the time of autopsy are shown. (e-h) Groups of lacZ-immunized C57BL/6J mice (n = 4) were injected either with α-Irr (white bars) or α-PLT (black and red bars) 3 h before injection of RAd35. One group (red bars) also received TKK-PLTs 36 h later. Mice receiving NaCl (dark gray bars) or α-PLT and TKK-PLTs but no RAd35 (light gray bars) served as controls. All animals were killed simultaneously, 3 d after RAd35 infection. Serum ALT (e), absolute numbers of intrahepatic CTLs (f) and Ifng mRNA (g) are expressed as mean ± standard deviation. (h) The platelet counts (mean ± standard deviation) in blood samples collected from all the animals in each group at the time of autopsy are shown.
Supplementary Figure 5. Platelet-dependent fibrin deposition within the inflamed liver. Immunohistochemical analysis of perfused livers from the same mice described in Supplementary Figure 4 using an Ab directed against human fibrinogen/fibrin (see Supplementary Methods online). Treatments were as follows: (a) NaCl, (b) α-Irr + CTL, (c) α-PLT + CTL and (d) α-PLT + CTL + TKK PLT, (e) NaCl, (f) α-Irr + RAd35, (g) α-PLT + RAd35 and (h) α-PLT + RAd35 + TKK-PLT. Scale bars, 50 μm (upper panels) and 150 μm (lower panels). Bottom right insets: mean serum ALT values at the time of autopsy.
Supplementary Table 1 α-PLT treatment selectively depletes platelet in vivo
We are grateful to S. Wieland for providing the pCMV-lacZ plasmid and for discussions. We also thank S. Medrano and M. Chadwell for technical assistance. This work was supported by US National Institutes of Health grants HL31950, HL42846, HL78784 (to Z.M.R.), CA40489 (to F.V.C.) and AI40696 (to L.G.G.). Work in the Board of Governors Gene Therapy Research Institute is funded by the Board of Governors at Cedars-Sinai Medical Center, the US National Institutes of Health and the Bram and Elaine Goldsmith Chair in Gene Therapeutics. This is manuscript number 16807-MEM from the Scripps Research Institute.
Note: Supplementary information is available on the Nature Medicine website.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.