RESUMEN
OBJECTIVE: Identifying components of immuneparesis, a hallmark of chronic liver failure, is crucial for our understanding of complications in cirrhosis. Various suppressor CD4+ T cells have been established as potent inhibitors of systemic immune activation. Here, we establish the presence, regulation and mechanism of action of a suppressive CD4+ T cell subset expressing human leucocyte antigen G (HLA-G) in patients with acute decompensation of cirrhosis (AD). DESIGN: Flow cytometry was used to determine the proportion and immunophenotype of CD4+HLA-G+ T cells from peripheral blood of 20 healthy controls (HCs) and 98 patients with cirrhosis (28 with stable cirrhosis (SC), 20 with chronic decompensated cirrhosis (CD) and 50 with AD). Transcriptional and functional signatures of cell-sorted CD4+HLA-G+ cells were delineated by NanoString technology and suppression assays, respectively. The role of immunosuppressive cytokine interleukin (IL)-35 in inducing this population was investigated through in vitro blockade experiments. Immunohistochemistry (IHC) and cultures of primary human Kupffer cells (KCs) were performed to assess cellular sources of IL-35. HLA-G-mediated T cell suppression was explored using neutralising antibodies targeting co-inhibitory pathways. RESULTS: Patients with AD were distinguished by an expansion of a CD4+HLA-G+CTLA-4+IL-35+ immunosuppressive population associated with disease severity, clinical course of AD, infectious complications and poor outcome. Transcriptomic analyses excluded the possibility that these were thymic-derived regulatory T cells. IHC analyses and in vitro cultures demonstrate that KCs represent a potent source of IL-35 which can induce the observed HLA-G+ phenotype. These exert cytotoxic T lymphocyte antigen-4-mediated impaired responses in T cells paralleled by an HLA-G-driven downregulation of T helper 17-related cytokines. CONCLUSION: We have identified a cytokine-driven peripherally derived suppressive population that may contribute to immuneparesis in AD.
Asunto(s)
Antígenos HLA-G , Subgrupos de Linfocitos T , Linfocitos T CD4-Positivos , Citocinas/metabolismo , Humanos , Interleucinas , Cirrosis Hepática/patologíaRESUMEN
OBJECTIVE: Immune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR- myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses. DESIGN: Patients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15-CD11b+HLA-DR- cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques. RESULTS: Circulating CD14+CD15-CD11b+HLA-DR- M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo. CONCLUSION: Immunosuppressive CD14+HLA-DR- M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent.
Asunto(s)
Insuficiencia Hepática Crónica Agudizada/inmunología , Antiinfecciosos/uso terapéutico , Tolerancia Inmunológica/inmunología , Células Supresoras de Origen Mieloide/inmunología , Adulto , Citocinas/metabolismo , Citometría de Flujo , Fucosiltransferasas/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Inmunofenotipificación , Antígeno Lewis X/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Fagocitosis/inmunología , Reacción en Cadena de la Polimerasa , PronósticoRESUMEN
UNLABELLED: Acetaminophen-induced acute liver failure (AALF) is characterized both by activation of innate immune responses and susceptibility to sepsis. Circulating monocytes and hepatic macrophages are central mediators of inflammatory responses and tissue repair processes during human AALF. Secretory leukocyte protease inhibitor (SLPI) modulates monocyte/macrophage function through inhibition of nuclear factor kappa B (NF-κB) signaling. The aims of this study were to establish the role of SLPI in AALF. Circulating levels of SLPI, monocyte cluster of differentiation 163 (CD163), human leukocyte antigen-DR (HLA-DR), and lipopolysaccharide (LPS)-stimulated levels of NF-κBp65, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 were determined in patients with AALF, chronic liver disease, and healthy controls. Immunohistochemistry and multispectral imaging of AALF explant tissue determined the cellular sources of SLPI and hepatic macrophage phenotype. The phenotype and function of monocytes and macrophages was determined following culture with recombinant human (rh)-SLPI, liver homogenates, and plasma derived from AALF patients in the presence and absence of antihuman (α)SLPI. Hepatic and circulatory concentrations of SLPI were elevated in AALF and immunohistochemistry revealed SLPI expression in biliary epithelial cells and within hepatic macrophages (h-mψ) in areas of necrosis. H-mψ and circulating monocytes in AALF exhibited an anti-inflammatory phenotype and functional characteristics; typified by reductions in NF-κBp65, TNF-α, and IL-6 and preserved IL-10 secretion following LPS challenge. Culture of healthy monocytes with AALF liver homogenates, plasma, or rhSLPI induced monocytes with strikingly similar anti-inflammatory characteristics which were reversed by inhibiting the activity of SLPI. CONCLUSION: SLPI is a pivotal mediator of anti-inflammatory responses in AALF through modulation of monocyte/macrophage function, which may account for the susceptibility to sepsis in AALF.
Asunto(s)
Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Inflamación/prevención & control , Inflamación/fisiopatología , Macrófagos/fisiología , Monocitos/fisiología , Inhibidor Secretorio de Peptidasas Leucocitarias/fisiología , Adolescente , Adulto , Antígenos CD/sangre , Antígenos de Diferenciación Mielomonocítica/sangre , Estudios de Casos y Controles , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Antígenos HLA-DR/sangre , Humanos , Inflamación/sangre , Interleucina-6/sangre , Persona de Mediana Edad , FN-kappa B/sangre , Fenotipo , Receptores de Superficie Celular/sangre , Inhibidor Secretorio de Peptidasas Leucocitarias/sangre , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/sangre , Adulto JovenRESUMEN
Acute liver failure (ALF) is a condition with a high mortality and morbidity for which new treatments are desperately required. We contend that although the initial event in ALF is liver cell death, the clinical syndrome of ALF and its complications including multi-organ dysfunction and sepsis, are largely generated by the immune response to liver injury. Hepatic macrophages fulfil a diversity of roles in ALF, from pro-inflammatory to pro-resolution. Their inherent plasticity means the same macrophages may have a variety of functions depending on the local tissue environment at different stages of disease. A better understanding of the mechanisms that regulate macrophage plasticity during ALF will be an essential step towards realising the potential of immune-modulating therapies that re-orientate macrophages to promote the desirable functions of attenuating liver injury and promoting liver repair/regenerative responses. The key dynamics: temporal (early vs. late phase), regional (hepatic vs. systemic), and activation (pro-inflammatory vs. pro-resolution) are discussed and the potential for novel ALF therapies that modulate monocyte/macrophage function are described.
Asunto(s)
Fallo Hepático Agudo/inmunología , Macrófagos/fisiología , Monocitos/fisiología , Animales , Polaridad Celular , Microambiente Celular , Humanos , Inmunidad Innata , Fallo Hepático Agudo/terapia , Regeneración Hepática , Síndrome de Respuesta Inflamatoria Sistémica/inmunologíaRESUMEN
UNLABELLED: Acetaminophen-induced acute liver failure (AALF) is associated with innate immunity activation, which contributes to the severity of hepatic injury and clinical outcome. A marked increase in hepatic macrophages (h-mφ) is observed in experimental models of AALF, but controversy exists regarding their role, implicating h-mφ in both aggravation and resolution of liver injury. The role of h-mφ in human AALF is virtually unexplored. We sought to investigate the role of chemokine (C-C motif) ligand 2 (CCL2) in the recruitment of circulating monocytes to the inflamed liver and to determine how the h-mφ infiltrate and liver microenvironment may contribute to tissue repair versus inflammation in AALF. We evaluated circulating monocytes, their chemokine (C-C motif) receptor 2 (CCR2) expression, and serum CCL2 levels in patients with AALF. Cell subsets and numbers of circulation-derived (MAC387+) or resident proliferating (CD68/Ki67+) h-mφ in hepatic immune infiltrates were determined by immunohistochemistry. Inflammatory cytokine levels were determined in whole and laser microdissected liver tissue by proteome array. In AALF, circulating monocytes were depleted, with the lowest levels observed in patients with adverse outcomes. CCL2 levels were high in AALF serum and hepatic tissue, and circulating monocyte subsets expressed CCR2, suggesting CCL2-dependent hepatic monocyte recruitment. Significant numbers of both MAC387+ and CD68+ h-mφ were found in AALF compared with control liver tissue with a high proportion expressing the proliferation marker Ki67. Levels of CCL2, CCL3, interleukin (IL)-6, IL-10, and transforming growth factor-ß1 were significantly elevated in AALF liver tissue relative to chronic liver disease controls. CONCLUSION: In AALF, the h-mφ population is expanded in areas of necrosis, both through proliferation of resident cells and CCL2-dependent recruitment of circulating monocytes. The presence of h-mφ within an anti-inflammatory/regenerative microenvironment indicates that they are implicated in resolution of inflammation/tissue repair processes during AALF.
Asunto(s)
Acetaminofén/envenenamiento , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/patología , Macrófagos/patología , Adulto , Analgésicos no Narcóticos/envenenamiento , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Quimiotaxis/inmunología , Femenino , Humanos , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Antígeno Ki-67/metabolismo , Fallo Hepático Agudo/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patología , Receptores CCR2/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Acute liver failure (ALF) is a devastating clinical syndrome characterised by progressive encephalopathy, coagulopathy, and circulatory dysfunction, which commonly leads to multiorgan failure and death. Central to the pathogenesis of ALF is activation of the immune system with mobilisation of cellular effectors and massive production of cytokines. As key components of the innate immune system, monocytes and macrophages are postulated to play a central role in the initiation, progression and resolution of ALF. ALF in humans follows a rapidly progressive clinical course that poses inherent difficulties in delineating the role of these pivotal immune cells. Therefore, a number of experimental models have been used to study the pathogenesis of ALF. Here we consider the evidence from experimental and human studies of ALF on the role of monocytes and macrophages in acute hepatic injury and the ensuing extrahepatic manifestations, including functional monocyte deactivation and multiple organ failure.
Asunto(s)
Fallo Hepático/sangre , Macrófagos/citología , Monocitos/citología , Animales , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Humanos , Sistema Inmunológico , Inflamación , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Fallo Hepático/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , RatasRESUMEN
Acute liver failure (ALF) shares striking similarities with septic shock with regard to the features of systemic inflammation, progression to multiple organ dysfunction and functional immunoparesis. While the existence of opposing systemic pro- and anti-inflammatory profiles resulting in organ failure and immune dysfunction are well recognised in septic shock, characterization of these processes in ALF has only recently been described. This review explores the evolution of the systemic inflammation in acute liver failure, its relation to disease progression, exacerbation of liver injury and development of innate immune dysfunction and extra-hepatic organ failure as sequelae. Defects in innate immunity are described in hepatic and extra-hepatic compartments. Clinical studies measuring levels of pro- and anti-inflammatory cytokines and expression of the antigen presentation molecule HLA-DR on monocytes, in combination with ex-vivo experiments, demonstrate that the persistence of a compensatory anti-inflammatory response syndrome, leading to functional monocyte deactivation, is a central event in the evolution of systemic immune dysfunction. Accurate immune profiling in ALF may permit the development of immunomodulatory strategies in order to improve outcome in this condition.
Asunto(s)
Fallo Hepático Agudo/inmunología , Apoptosis , Proteínas del Sistema Complemento/metabolismo , Citocinas/metabolismo , Fibronectinas/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Inmunidad Innata , Técnicas In Vitro , Células Asesinas Naturales/inmunología , Macrófagos del Hígado/inmunología , Fallo Hepático Agudo/etiología , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/terapia , Macrófagos/inmunología , Modelos Inmunológicos , Monocitos/inmunología , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/inmunología , Células T Asesinas Naturales/inmunología , Neutrófilos/inmunología , Pronóstico , Sepsis/etiología , Sepsis/inmunología , Transducción de Señal , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/inmunologíaRESUMEN
Acute liver failure (ALF) shares striking similarities with septic shock where a decrease in HLA-DR expression on monocytes is associated with disease severity and predicts outcome. We investigated monocyte HLA-DR expression in ALF in relation to inflammatory mediator levels and clinical outcome. Monocyte HLA-DR expression was determined in 50 patients with acetaminophen-induced ALF (AALF) and 20 non-acetaminophen-induced ALF (NAALF). AALF patients were divided into dead/transplanted (AALF-NS, n = 26) and spontaneous survivors (AALF-S, n = 24). Fifty patients with chronic liver disease (CLD) and 50 healthy volunteers served as controls. Monocyte HLA-DR expression was determined by double-color flow-cytometry with monoclonal antibodies detecting HLA-DR and monocyte specific CD14. Serum levels of interleukin (IL) -4, -6, -10, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were concomitantly measured by ELISA. Compared to healthy volunteers (75%) and CLD (67%) monocyte HLA-DR percentage expression was lower in AALF (15%, P < .001) and NAALF (22 %, P < .001). Compared to AALF-S, AALF-NS had lower monocyte HLA-DR % (11% vs. 36%, P < .001) and higher levels of IL-4, IL-6, IL-10 and TNF-alpha (P < .001). HLA-DR percentage negatively correlated with INR, blood lactate, pH and levels of encephalopathy (r = -0.8 to -0.5, P < .01), IL-10 (r = -0.8, P < .0001), TNF-alpha (r = -0.4, P = .02). HLA-DR percentage level Asunto(s)
Acetaminofén/efectos adversos
, Analgésicos no Narcóticos/efectos adversos
, Antígenos HLA-DR/biosíntesis
, Fallo Hepático Agudo/metabolismo
, Monocitos/metabolismo
, Adulto
, Biomarcadores/metabolismo
, Citocinas/sangre
, Ensayo de Inmunoadsorción Enzimática
, Femenino
, Citometría de Flujo
, Humanos
, Fallo Hepático Agudo/inducido químicamente
, Masculino
, Persona de Mediana Edad
, Pronóstico
, Índice de Severidad de la Enfermedad