RESUMO
Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely. Approximately 60% of these recipient DCs displayed donor MHC class I, indicating cross-dressing. By contrast, only a very minor fraction (0%-2%) of cross-dressed DCs (CD-DCs) was evident in the spleen. CD-DCs sorted from liver grafts expressed much higher levels of T cell inhibitory programed death ligand 1 (PD-L1) and high levels of interleukin-10 compared with non-CD-DCs (nCD-DCs) isolated from the graft. Concomitantly, high incidences of programed death protein 1 (PD-1)hi T cell immunoglobulin and mucin domain containing 3 (TIM-3)+ exhausted graft-infiltrating CD8+ T cells were observed. Unlike nCD-DCs, the CD-DCs failed to stimulate proliferation of allogeneic T cells but markedly suppressed antidonor host T cell proliferation. CD-DCs were much less evident in allografts from DNAX-activating protein of 12 kDa (DAP12)-/- donors that were rejected acutely. CONCLUSION: These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).
Assuntos
Células Dendríticas/imunologia , Sobrevivência de Enxerto/imunologia , Fígado/imunologia , Tolerância ao Transplante/imunologia , Animais , Citometria de Fluxo , Microscopia Intravital , Transplante de Fígado/efeitos adversos , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
BACKGROUND: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection. METHODS: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts. RESULTS: The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal. CONCLUSIONS: Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.
Assuntos
Trifosfato de Adenosina/química , Rejeição de Enxerto , Transplante de Coração , Sistema Imunitário/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Morfolinas/farmacologia , Animais , Benzamidas , Proliferação de Células , Células Dendríticas/citologia , Sobrevivência de Enxerto/efeitos dos fármacos , Imunoglobulina G/química , Imunossupressores/farmacologia , Masculino , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas , Sirolimo/farmacologia , Linfócitos T/citologia , Transplante HomólogoRESUMO
The surgically demanding mouse orthotopic liver transplant model was first described in 1991. It has proved to be a powerful research tool for the investigation of liver biology, tissue injury, the regulation of alloimmunity and tolerance induction, and the pathogenesis of specific liver diseases. Liver transplantation in mice has unique advantages over transplantation of the liver in larger species, such as the rat or pig, because the mouse genome is well characterized and there is much greater availability of both genetically modified animals and research reagents. Liver transplant experiments using various transgenic or gene knockout mice have provided valuable mechanistic insights into the immunobiology and pathobiology of the liver and the regulation of graft rejection and tolerance over the past 25 years. The molecular pathways identified in the regulation of tissue injury and promotion of liver transplant tolerance provide new potential targets for therapeutic intervention to control adverse inflammatory responses/immune-mediated events in the hepatic environment and systemically. In conclusion, orthotopic liver transplantation in the mouse is a valuable model for gaining improved insights into liver biology, immunopathology, and allograft tolerance that may result in therapeutic innovation in the liver and in the treatment of other diseases.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Fígado/veterinária , Fígado/imunologia , Traumatismo por Reperfusão/veterinária , Tolerância ao Transplante , Animais , Rejeição de Enxerto/veterinária , Células-Tronco Hematopoéticas/imunologia , Fígado/fisiopatologia , Fígado/cirurgia , Hepatopatias/etiologia , Transplante de Fígado/métodos , Camundongos , Modelos Animais , Traumatismo por Reperfusão/metabolismo , Transdução de SinaisRESUMO
UNLABELLED: Plasmacytoid dendritic cells (pDC) constitute the body's principal source of type I interferon (IFN) and are comparatively abundant in the liver. Among various cytokines implicated in liver ischemia and reperfusion (I/R) injury, type I IFNs have been described recently as playing an essential role in its pathogenesis. Moreover, type I IFNs have been shown to up-regulate hepatocyte expression of IFN regulatory factor 1 (IRF-1), a key transcription factor that regulates apoptosis and induces liver damage after I/R. Our aim was to ascertain the capacity of IFN-α released by liver pDC to induce liver damage through hepatic IRF-1 up-regulation after I/R injury. Our findings show that liver pDC mature and produce IFN-α in response to liver I/R. Liver pDC isolated after I/R induced elevated levels of IRF-1 production by hepatocytes compared with liver pDC isolated from sham-operated mice. Notably, hepatic IRF-1 expression was reduced significantly by neutralizing IFN-α. In vivo, IFN-α neutralization protected the liver from I/R injury by reducing hepatocyte apoptosis. This was associated with impaired expression of IRF-1 and proapoptotic molecules such as Fas ligand, its receptor (Fas) and death receptor 5, which are regulated by IRF-1. Furthermore, pDC-depleted mice failed to up-regulate hepatic IFN-α and displayed less liver injury associated with reduced levels of hepatic interleukin (IL)-6, tumor necrosis factor-α, and hepatocyte apoptosis after I/R compared with controls. CONCLUSION: these data support the hypothesis that IFN-α derived from liver pDC plays a key role in the pathogenesis of liver I/R injury by enhancing apoptosis as a consequence of induction of hepatocyte IRF-1 expression.
Assuntos
Células Dendríticas/imunologia , Fator Regulador 1 de Interferon/imunologia , Interferon-alfa/imunologia , Hepatopatias/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Apoptose/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Hepatócitos/citologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Interferon-alfa/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/patologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/imunologiaRESUMO
UNLABELLED: Hepatic innate immune cells, in particular, interstitial dendritic cells (DCs), regulate inflammatory responses and may promote inherent liver tolerogenicity. After tissue injury, adenosine triphosphate (ATP) is released and acts as a damage-associated molecular pattern that activates innate immune cells by pattern recognition receptors. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) rapidly hydrolyzes extracellular ATP to maintain physiological levels. We hypothesized that CD39 expression on liver DCs might contribute to regulation of their innate immune functions. Mouse liver conventional myeloid DCs (mDCs) were hyporesponsive to ATP, compared with their splenic counterparts. This disparity was ascribed to more efficient hydrolysis of ATP by higher expression of CD39 on liver mDCs. Human liver mDCs expressed greater levels of CD39 than those from peripheral blood. The comparatively high expression of CD39 on liver mDCs correlated strongly with both ATP hydrolysis and adenosine production. Notably, CD39(-/-) mouse liver mDCs exhibited a more mature phenotype, greater responsiveness to Toll-like receptor 4 ligation, and stronger proinflammatory and immunostimulatory activity than wild-type (WT) liver mDCs. To investigate the role of CD39 on liver mDCs in vivo, we performed orthotopic liver transplantation with extended cold preservation using CD39(-/-) or WT donor mouse livers. Compared to WT liver grafts, CD39(-/-) grafts exhibited enhanced interstitial DC activation, elevated proinflammatory cytokine levels, and more-severe tissue injury. Moreover, portal venous delivery of WT, but not CD39(-/-) liver mDCs, to donor livers immediately post-transplant exerted a protective effect against graft injury in CD39(-/-) to CD39(-/-) liver transplantation. CONCLUSIONS: These data reveal that CD39 expression on conventional liver mDCs limits their proinflammatory activity and confers protective properties on these important innate immune cells against liver transplant ischemia/reperfusion injury.