Patrick Bertolino

TLR4 activation mediates kidney ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4(-/-) and MyD88(-/-) mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4(-/-) and MyD88(-/-) TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4(-/-) mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4(-/-) BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.

The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity

Hepatic immunobiology is paradoxical: although the liver possesses unusual tolerogenic properties, it is also the site of effective immune responses against multiple pathogens and subject to immune-mediated pathology. The mechanisms underlying this dichotomy remain unclear. Following previous work demonstrating that the liver may act as a site of primary T cell activation, we demonstrate here that the balance between immunity and tolerance in this organ is established by competition for primary activation of CD8+ T cells between the liver and secondary lymphoid tissues, with the immune outcome determined by the initial site of activation. Using a transgenic mouse model in which antigen is expressed within both liver and lymph nodes, we show that while naive CD8+ T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury. The implications of these novel findings may pertain not only to the normal maintenance of peripheral tolerance, but also to hepatic allograft tolerance and the immunopathogenesis of chronic viral hepatitis.

T lymphocytes interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells

The liver has an established ability to induce tolerance. Recent evidence indicates that this unique property might be related to its distinctive architecture allowing T cells to be activated in situ independently of lymphoid tissues. Unlike lymph node–activated T cells, liver‐activated T cells are short‐lived, a mechanism that might contribute to the “liver tolerance effect.” Although the potential role of hepatocytes as tolerogenic antigen‐presenting cells has been demonstrated, the question as to whether these cells are able to interact with CD8+ T cells in physiological settings remains controversial. Contradicting the immunological dogma stating that naïve T lymphocytes are prevented from interacting with parenchymal cells within non‐lymphoid organs by an impenetrable endothelial barrier, we show here that the unique morphology of the liver sinusoidal endothelial cell (LSEC) permits interactions between lymphocytes and hepatocytes. Using electron microscopy, we demonstrate that liver resident lymphocytes as well as circulating naïve CD8+ T cells make direct contact with hepatocytes through cytoplasmic extensions penetrating the endothelial fenestrations that perforate the LSECs. Furthermore, the expression of molecules required for primary T cell activation, MHC class I and ICAM‐1, is polarized on hepatocytes to the perisinusoidal cell membrane, thus maximizing the opportunity for interactions with circulating lymphocytes. In conclusion, this study has identified, at the ultrastructural level, a unique type of interaction between naïve T lymphocytes and liver parenchymal cells in vivo. These results hold implications for the pathogenesis of viral hepatitis in which hepatocytes may represent the main antigen‐presenting cell, and for the development of immune tolerance as lymphocytes pass through the liver. (HEPATOLOGY 2006;44:1182–1190.)

Autocrine IL-10 impairs dendritic cell (DC)-derived immune responses to mycobacterial infection by suppressing DC trafficking to draining lymph nodes and local IL-12 production

The production of IL‐12 by dendritic cells (DC) early in an immune response is considered critical for the polarization of CD4+ T lymphocyte response towards a Th1 pattern, a key process in the clearance of intracellular pathogens. Infection of bone marrow‐derived DC with Mycobacterium bovis Bacillus Calmette Guérin (BCG) induced a concurrent and dose‐dependent releaseof IL‐10 and IL‐12. Here we examined whether the production of IL‐10 by DC affected their IL‐12 response to mycobacterial infection and the generation of protective immune responses in vivo. Compared to wild‐type (WT) DC, DC deficient for IL‐10 synthesis (IL‐10–/–) showed increased IL‐12 production in response to BCG infection and CD40 stimuli in vitro. Moreover, when transferred into mice, infected IL‐10–/– DC were more efficient than WT DC at inducing IFN‐γ production to mycobacterial antigens in the draining lymph nodes (DLN).This effect was associated with increased trafficking of IL‐10–/– DC to the DLN and enhanced IL‐12 production by DC within the DLN. These data show that autocrine IL‐10 exerts a dual inhibitory effect on the induction of primary immune responses by DC: first, by down‐regulating the migration of infected DC to the DLN and second, by modulating the IL‐12 production by DC in the DLN.

Antigen-Specific Primary Activation of CD8+ T Cells Within the Liver

It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8+ T cells specific for H-2Kb were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-Kb mice expressing H-2Kb in the liver. Activated CD8+ cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8+ cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8+ T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8+ T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis.

Role of primary intrahepatic T-cell activation in the 'liver tolerance effect'

There is accumulating evidence suggesting that hepatic permeability to both naive and activated T lymphocytes may be unique among the solid organs. The possibility that the liver may act as a site of primary activation for CD8+ T lymphocytes is supported by experimental data and may contribute to some of the unique immunological properties of this organ, particularly its ability to induce antigen‐specific tolerance. This review discusses the nature of the liver APC inducing primary T‐cell activation within the liver: Kupffer cells, liver dendritic cells, liver sinusoidal endothelial cells and hepatocytes are favourably located to allow physical contact with circulating T lymphocytes. Here, we examine the capability of each cell type to act as APC for naive CD4+ or CD8+ T cells and to induce tolerance.

Intrahepatic Murine CD8 T-Cell Activation Associates With a Distinct Phenotype Leading to Bim-Dependent Death

BACKGROUND & AIMS Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo. METHODS Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naïve CD8 T cells specific for the hepatic antigen. RESULTS Liver-activated CD8 T cells displayed poor effector functions and a unique CD25(low) CD54(low) phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation. CONCLUSIONS This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.

Hepatocyte entry leads to degradation of autoreactive CD8 T cells

Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this “suicidal emperipolesis” is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.

Toward MSC in Solid Organ Transplantation: 2008 Position Paper of the MISOT Study Group

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance—agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.

Cell‐mediated rejection results in allograft loss after liver cell transplantation

Liver cell transplantation in humans has been impeded by invariable loss of the graft. It is unclear whether graft loss is due to an immune response against donor hepatocytes. Transplantation with ABO‐matched liver cells was performed in a patient with Crigler‐Najjar type 1. After successful engraftment, there was a gradual loss of graft function. Solid‐phase enzyme immunoassay testing and cell‐complement cytotoxicity assays detecting preformed antibodies directed toward class I and/or class II human leukocyte antigen (HLA) molecules were negative. In contrast, a striking host alloresponse to either the HLA‐B39 or C7 antigen was found, suggesting that a vigorous response to a defined mismatched HLA antigen contributed to graft loss in our patient. This study provides evidence that a T‐cell–mediated immune mechanism could be responsible for human liver cell transplant graft loss. This finding warrants confirmation in future liver cell transplants in humans. Liver Transpl 14:688–694, 2008.