Maria Cecilia S. Freitas

The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes.

Background Anti–HLA-DQ antibodies are the predominant HLA class II donor-specific antibodies (DSAs) after transplantation. Recently, de novo DQ DSA has been associated with worse allograft outcomes. The aim of this study was to determine the further complement-binding characteristics of the most harmful DQ DSA. Methods Single-antigen bead technology was used to screen 284 primary kidney transplant recipients for the presence of posttransplantation DQ DSA. Peak DSA sera of 34 recipients with only de novo DQ DSA and of 20 recipients with de novo DQ plus other DSAs were further analyzed by a modified single-antigen bead assay using immunoglobulin (Ig)-G subclass-specific reporter antibodies and a C1q-binding assay. Results Compared with recipients who did not have DSA, those with de novo persistent DQ-only DSA and with de novo DQ plus other DSAs had more acute rejection (AR) episodes (22%, P=0.005; and 36%, P=0.0009), increased risk of allograft loss (hazards ratio, 3.7, P=0.03; and hazards ratio, 11.4, P=0.001), and a lower 5-year allograft survival. De novo DQ-only recipients with AR had more IgG1/IgG3 combination and C1q-binding antibodies (51%, P=0.01; and 63%, P=0.001) than patients with no AR. Furthermore, the presence of C1q-binding de novo DQ DSA was associated with a 30% lower 5-year allograft survival (P=0.003). Conclusions The presence of de novo persistent, complement-binding DQ DSA negatively impacts kidney allograft outcomes. Therefore, early posttransplantation detection, monitoring, and removal of complement-binding DQ might be crucial for improving long-term kidney transplantation outcomes.

CXCL10 regulates liver innate immune response against ischemia and reperfusion injury

We have shown that activation of toll‐like receptor 4 (TLR4) and its interferon regulatory factor 3 (IRF3)‐dependent downstream signaling pathway are required for the development of liver ischemia/reperfusion injury (IRI). This study focused on the role of TLR4‐IRF3 activation pathway products, in particular, chemokine (C‐X‐C motif) ligand 10 (CXCL10). The induction of CXCL10 by liver IR was rapid (1 hour postreperfusion), restricted (ischemic lobes), and specific (no CXCL9 and CXCL11 induction). Functionally, CXCL10 was critical for IR‐induced liver inflammation and hepatocellular injury. CXCL10 knockout (KO) mice were protected from IRI, as evidenced by reduced serum alanine aminotransferase (sALT) levels and preserved liver histological detail. The induction of pro‐inflammatory genes, such as tumor necrosis factor alpha (TNF‐α), interleukin 1β (IL‐1β), IL‐6, and IL‐12β was diminished, whereas the induction of the IL‐10 gene remained intact in CXCL10 KO mice, indicating an altered liver response against IR. This was accompanied by selective down‐regulation of extracellular signal‐regulated kinase (ERK), but intact Jun N‐terminal kinase (JNK), activation in the KO IR livers. This altered liver inflammation response was (1) specific to IR, because lipopolysaccharide (LPS) induced a comparable pro‐inflammatory response in CXCL10 KO and wild‐type (WT) mice; and (2) responsible for liver cytoprotection from IR, because neutralization of IL‐10 restored local inflammation and hepatocellular damage. Conclusion: CXCL10 regulates liver inflammation response against IRI, and its deficiency protected livers from IRI by local IL‐10–mediated cytoprotection. Targeting CXCL10 may provide a novel therapeutic means to ameliorate liver IRI in clinics. (HEPATOLOGY 2008.)

T-Cell Immunoglobulin Mucin-3 Determines Severity of Liver Ischemia/Reperfusion Injury in Mice in a TLR4-Dependent Manner

BACKGROUND & AIMS T-cell immunoglobulin mucin (TIM) genes are expressed by T cells and regulate host immunity and tolerance. CD4(+) T cells mediate innate immunity-dominated liver ischemia-reperfusion injury (IRI) by unknown mechanisms. TIM-1 is involved in liver IRI, which is activated in part by the Toll-like receptor (TLR)4; we investigated the role of TIM-3 and TLR4 in IRI. METHODS Using an antibody against TIM-3 (anti-TIM-3), we studied TIM-3 signaling in mice following partial warm liver ischemia and reperfusion. RESULTS Mice given anti-TIM-3 had more liver damage than controls. Histological studies revealed that anti-TIM-3 increased hepatocellular damage and local neutrophil infiltration, facilitated local accumulation of T cells and macrophages, and promoted liver cell apoptosis. Intrahepatic neutrophil activity; induction of proinflammatory cytokines and chemokines; and expression of cleaved caspase-3, nuclear factor-κB, and TLR4 all increased in mice given anti-TIM-3. Administration of anti-TIM-3 followed by anti-galectin-9 (Gal-9 is a TIM-3 ligand) increased production of interferon-γ by concanavalin A (ConA)-stimulated spleen T cells and expression of tumor necrosis factor-α and interleukin-6 in ConA-stimulated macrophages co-cultured with T cells. Anti-TIM-3 did not affect liver IRI in TLR4-deficient mice. CONCLUSION TIM-3 blockade exacerbated local inflammation and liver damage, indicating the importance of TIM-3-Gal-9 signaling in maintaining hepatic homeostasis. TIM-3-TLR4 cross-regulation determined the severity of liver IRI in TLR4-dependent manner; these findings provide important information about the modulation of innate vs adaptive responses in patients that received liver transplants. Negative co-stimulation signaling by hepatic T-cells might be developed to minimize innate immunity-mediated liver tissue damage.

HO-1 - STAT3 Axis in Mouse Liver Ischemia/Reperfusion Injury: Regulation of TLR4 Innate Responses Through PI3K/PTEN Signaling

BACKGROUND & AIMS Signal transducer and activator of transcription 3 (STAT3), a key mediator of anti-inflammatory cytokine signaling, is essential for heme oxygenase-1 (HO-1)-induced cytoprotection. The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog delete on chromosome 10 (PTEN) pathways regulate diverse innate immune responses. This study was designed to investigate the role of STAT3 in the regulation of PI3K/PTEN cascade after HO-1 induction in a mouse model of innate immune-dominated liver ischemia/reperfusion injury (IRI). METHODS Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6h of reperfusion. RESULTS Mice subjected to Ad-HO-1 transfer were resistant to liver IRI, and this cytoprotective effect correlated with increased intrahepatic PI3K/Akt and diminished PTEN expression. In contrast, mice undergoing adjunctive Ad-HO-1 treatment and STAT3 knockdown (siRNA) remained susceptible to IR-mediated local inflammatory response and hepatocellular damage. Consistent with decreased cell apoptosis and inhibited TLR4 expression after PI3K/Akt activation, treatment with specific PI3k inhibitor increased local inflammation and recreated liver IRI despite Ad-HO-1 gene transfer. Parallel in vitro studies with bone marrow derived-macrophages have confirmed that HO-1-STAT3 axis-induced PI3K/Akt negatively regulated PTEN expression in TLR4-dependent fashion. CONCLUSIONS These findings underscore the role of HO-1 induced STAT3 in modulating PI3K/PTEN in liver IRI cascade. Activating PI3K/Akt provides negative feedback mechanism for TLR4-driven inflammation. Identifying molecular pathways of STAT3 modulation in the innate immune system provides the rationale for novel therapeutic approaches for the management of liver inflammation and IRI in transplant patients.

The emerging role of T cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse.

The T cell immunoglobulin and mucin domain‐containing molecules (TIM) protein family, which is expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity‐dominated organ ischemia–reperfusion injury (IRI), remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti–TIM‐1 antibody (Ab), we studied the role of TIM‐1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti–TIM‐1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared with controls. Histological examination has revealed that anti–TIM‐1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM‐1 ligand–expressing TIM‐4+ cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of proinflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in vitro studies, anti–TIM‐1 Ab suppressed interferon‐γ (IFN‐γ) production in concanavalin A (conA)–stimulated spleen T cells, and diminished tumor necrosis factor α (TNF‐α)/interleukin (IL)‐6 expression in a macrophage/spleen T cell coculture system. This is the first study to provide evidence for the novel role of TIM‐1 signaling in the mechanism of liver IRI. TIM‐1 regulates not only T for the role of cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM‐1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation. (HEPATOLOGY 2010.)

Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury.

Programmed death‐1 (PD‐1)/B7‐H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity‐dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD‐1/B7‐H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD‐1 signaling after anti–B7‐H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7‐H1 immunoglobulin (B7‐H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well‐preserved liver architecture. The therapeutic potential of B7‐H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti‐necrotic/apoptotic Bcl‐2/Bcl‐xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)‐10. Neutralization of IL‐10 re‐created liver IRI and rendered B7‐H1Ig–treated hosts susceptible to IRI. These findings were confirmed in T cell–macrophage in vitro coculture in which B7‐H1Ig diminished tumor necrosis factor‐α/IL‐6 levels in an IL‐10–dependent manner. Our novel findings document the essential role of the PD‐1/B7‐H1 pathway in liver IRI. Conclusion: This study is the first to demonstrate that stimulating PD‐1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD‐1 upon T cell–Kupffer cell cross‐talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL‐10–dependent cytoprotection. (HEPATOLOGY 2010.)

The Inhibition of Neutrophil Elastase Ameliorates Mouse Liver Damage Due to Ischemia and Reperfusion

Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS‐D chloroacetate esterase stains, and immunohistochemistry (anti–Ly‐6G). The expression of pro‐inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C‐X‐C motif) ligand 1 (CXCL‐1), CXCL‐2, and CXCL‐10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining and caspase‐3 activity. In addition, toll‐like receptor 4 (TLR4) expression was diminished in NEI‐pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939–947, 2009.

The membrane attack complex (C5b‐9) in liver cold ischemia and reperfusion injury

Activation of the complement cascade represents an important event during ischemia/reperfusion injury (IRI). This work was designed to investigate the role of the membrane attack complex (MAC; C5b‐9) in the pathogenesis of hepatic IRI. Livers from B&W/Stahl/rC6(+) and C6(−) rats were harvested, stored for 24 hours at 4°C, and then transplanted [orthotopic liver transplantation (OLT)] to syngeneic recipients. There were 4 experimental groups: (1) C6(+)→C6(+), (2) C6(+)→C6(−), (3) C6(−)→C6(+), and (4) C6(−)→C6(−). At day +1, C6(−) OLTs showed decreased vascular congestion/necrosis, contrasting with extensive necrosis in C6(+) livers, that was independent of the recipient C6 status (Suzuki score: 7.2 ± 0.9, 7.3 ± 1.3, 4.5 ± 0.6, and 4.8 ± 0.4 for groups 1‐4, respectively, P < 0.05). The liver function improved in recipients of C6(−) grafts (serum glutamic oxaloacetic transaminase: 2573 ± 488, 1808 ± 302, 1170 ± 111, and 1188 ± 184 in groups 1‐4, respectively, P < 0.05). Intragraft macrophage infiltration (ED‐1 immunostaining) and neutrophil infiltration (myeloperoxidase activity) were reduced in C6(−) grafts versus C6(+) grafts (P = 0.001); these data were confirmed by esterase staining (naphthol). The expression of proinflammatory interferon‐γ, interleukin‐1β, and tumor necrosis factor messenger RNA/protein was also reduced in C6(−) OLTs in comparison with C6(+) OLTs. Western blot–assisted expression of proapoptotic caspase‐3 was decreased in C6(−) OLTs versus C6(+) OLTs (P = 0.006), whereas antiapoptotic Bcl‐2/Bag‐1 was enhanced in C6(−) OLTs compared with C6(+) OLTs (P = 0.001). Terminal deoxynucleotidyl transferase–mediated dUTP nick end‐labeling staining of apoptotic cells was enhanced (P < 0.05) in C6(+) OLTs compared with C6(−) OLTs. Thus, the terminal products of the complement system are essential in the mechanism of hepatic IRI. This is the first report using a clinically relevant liver cold ischemia model to show that local MAC inhibition attenuates IRI cascade in OLT recipients. Liver Transpl 14:1133–1141, 2008.

The Protective Function of Neutrophil Elastase Inhibitor in Liver Ischemia/Reperfusion Injury

Background. A neutrophil elastase (NE) inhibitor, Sivelestat, has been approved for the treatment of acute lung injury associated with systemic inflammation in humans. Some reports have also shown its protective effects in liver inflammatory states. We have recently documented the importance of NE in the pathophysiology of liver ischemia/reperfusion injury, a local Ag-independent inflammation response. This study was designed to explore putative cytoprotective functions of clinically available Sivelestat in liver ischemia/reperfusion injury. Methods. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6 or 24 hr of reperfusion. The mice were given Sivelestat (100 mg/kg, subcutaneous) at 10 min before ischemia, 10 min before reperfusion, and at 1 and 3 hr of reperfusion thereafter. Results. Sivelestat treatment significantly reduced serum alanine aminotransferase levels and NE activity, when compared with controls. Histological liver examination has revealed that unlike in controls, Sivelestat ameliorated the hepatocellular damage and decreased local neutrophil activity and infiltration. The expression of proinflammatory cytokines (tumor necrosis factor-&agr; and interleukin-6), chemokines (CXCL-1, CXCL-2, and CXCL-10), and toll-like receptor 4 was significantly reduced in the treatment group, along with diminished apoptosis through caspase-3 pathway. Moreover, in vitro studies confirmed downregulation of proinflammatory cytokine and chemokine programs in mouse macrophage cell cultures, along with depression of innate toll-like receptor 4 signaling. Conclusion. Sivelestat-mediated NE inhibition may represent an effective therapeutic option in liver transplantation and other inflammation disease states.

TYPE I INTERFERON PATHWAY MEDIATES RENAL ISCHEMIA/REPERFUSION INJURY

Background. Ischemia/reperfusion (I/R) injury is a common cause of acute renal failure after kidney transplantation. This study was designed to analyze the role of type I interferon (IFN) signaling downstream of Toll-like receptor 2/Toll-like receptor 4 activation in the mechanism of I/R-triggered kidney damage. Methods. Local warm ischemia was induced in groups wild-type (WT) and type I IFN receptor (IFNAR)−/− mice (C57BL/6) by clamping both kidney pedicles for 45 min. Mice were killed at 5/24/72 hr after reperfusion for serum and kidney sampling. Results. At 5 hr, serum creatinine and blood urea nitrogen levels were markedly reduced in IFNAR−/− mice as compared with WT. By 24 hr after reperfusion, both serum creatinine/blood urea nitrogen in WT increased further, whereas those in IFNAR−/− mice remained comparable with sham controls. Histological analyses showed significantly higher percentage of tubules in the outer medulla displaying cell necrosis, loss of the brush border, cast formation and tubular dilatation in WT mice, as compared with IFNAR−/−. Immunohistology revealed increased neutrophil and macrophage infiltration in the outer medulla in WT mice. The expression of proinflammatory tumor necrosis factor-&agr;, interleukin-1, interleukin-6, and CXCL-2 was markedly reduced selectively in IFNAR−/− mice. Finally, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling analysis showed significantly decreased frequency of apoptotic tubular epithelial cells in IFNAR-deficient mice, as compared with WT. Conclusion. This is the first report, which documents the key role of type I IFN signaling in the mechanism of kidney I/R injury. Type I IFN may thus serve as a novel target for the therapy against renal I/R injury.