Jaap Kwekkeboom

Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B

Dendritic cells (DC) play an important role in the induction of T‐cell responses. We hypothesize that the hampered antiviral T‐cell response in chronic hepatitis B patients is a result of impaired dendritic cell function. In this study, we compared the number, phenotype and functionality of two important blood precursor DC, myeloid DC (mDC) and plasmacytoid DC (pDC), of chronic hepatitis B patients with healthy volunteers. No differences in percentages of mDC and pDC in peripheral blood mononuclear cells were observed between chronic hepatitis B patients and healthy controls. The allostimulatory capacity of isolated and in vitro matured mDC, but not of pDC, was significantly decreased in patients compared to controls. Accordingly, a decreased percentage of mDC expressing CD80 and CD86 was observed after maturation, compared to controls. In addition, mDC of patients showed a reduced capacity to produce tumor necrosis factor α after a stimulus with synthetic double‐stranded RNA and interferon γ. Purified pDC from patients produced less interferon α, an important antiviral cytokine, in response to stimulation with Staphylococcus aureus Cowan strain I than pDC isolated from controls. In conclusion, mDC and pDC are functionally impaired in patients with chronic hepatitis B. This might be an important way by which hepatitis B virus evades an adequate immune response, leading to viral persistence and disease chronicity. (HEPATOLOGY 2004;40:738–746.)

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.

Modulation of the cellular immune system by intravenous immunoglobulin

Intravenous immunoglobulin (IVIg) is therapeutically used in a variety of immune-mediated diseases. The beneficial effects of IVIg in auto-antibody-mediated diseases can be explained by neutralization, accelerated clearance and prevention of Fcgamma-receptor binding of auto-antibodies. However, the means by which IVIg exerts therapeutic effects in disorders mediated by cellular immunity have remained enigmatic. Clinical improvements, followed by IVIg treatment, often extend beyond the half-life of infused IgG, thereby indicating that IVIg modifies the cellular immune compartment for a prolonged period. Here, we discuss recent advances in the understanding of different, mutually non-exclusive mechanisms of action of IVIg on cells of the innate and adaptive immune system. These mechanisms might explain the beneficial effects of IVIg in certain autoimmune and inflammatory diseases.

Hepatocyte‐derived microRNAs as serum biomarkers of hepatic injury and rejection after liver transplantation

Recent animal and human studies have highlighted the potential of hepatocyte‐derived microRNAs (HDmiRs) in serum as early, stable, sensitive, and specific biomarkers of liver injury. Their usefulness in human liver transplantation, however, has not been addressed. The aim of this study was to investigate serum HDmiRs as markers of hepatic injury and rejection in liver transplantation. Serum samples from healthy controls and liver transplant recipients (n = 107) and peritransplant liver allograft biopsy samples (n = 45) were analyzed via the real‐time polymerase chain reaction quantification of HDmiRs (miR‐122, miR‐148a, and miR‐194). The expression of miR‐122 and miR‐148a in liver tissue was significantly reduced with prolonged graft warm ischemia times. Conversely, the serum levels of these HDmiRs were elevated in patients with liver injury and positively correlated with aminotransferase levels. HDmiRs appear to be very sensitive because patients with normal aminotransferase values (<50 IU/L) had 6‐ to 17‐fold higher HDmiR levels in comparison with healthy controls (P < 0.005). During an episode of acute rejection, serum HDmiRs were elevated up to 20‐fold, and their levels appeared to rise earlier than aminotransferase levels. HDmiRs in serum were stable during repeated freezing and thawing. In conclusion, this study shows that liver injury is associated with the release of HDmiRs into the circulation. HDmiRs are promising candidates as early, stable, and sensitive biomarkers of rejection and hepatic injury after liver transplantation. Liver Transpl 18:290–297, 2012.

Low circulating regulatory T‐cell levels after acute rejection in liver transplantation

Immune regulatory CD4+CD25+ T cells play a crucial role in inducing and maintaining allograft tolerance in experimental models of transplantation (Tx). In humans, the effect of Tx and immunosuppression on the function and homeostasis of CD4+CD25+ regulatory T cells (Tregs) is not well characterized. In this study, the frequency of Tregs in liver transplant recipients was determined based on flow cytometric analysis of CD4, CD25, CD45RO, and cytotoxic T lymphocyte antigen (CTLA)‐4 markers, and the suppressor activity of Tregs was assessed in a mixed‐leukocyte reaction. A link between Tregs, acute rejection, and immune‐suppressive treatment was investigated. Liver transplant recipients had significantly higher Treg levels in peripheral blood pre‐Tx than healthy controls. After Tx, a significant drop in the Treg fraction was observed. This reduction of circulating Tregs was transient and was associated with immunosuppression. In recipients who did not develop rejection, a relative recovery of Treg levels was seen within the first year after Tx. Recipients who experienced an episode of steroid‐treated acute rejection, however, had sustained low Treg levels. The suppressive activities of CD4+CD25+ Tregs from rejectors, nonrejectors, and healthy controls on proliferation and interferon (IFN)‐γ production were indistinguishable. In conclusion, the percentage of CD4+CD25+CD45RO+CTLA‐4+ quadruple‐positive Tregs in peripheral blood decreases significantly after liver Tx. Treatment with methylprednisolone during Tx and for acute rejection is associated with low circulating Tregs. Despite these quantitative differences between rejectors and nonrejectors, the suppressive quality of CD4+CD25+ Tregs is identical in both groups. Liver Transpl 12:277–284, 2006.

Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

Background/aims RNA interference (RNAi), a sequence-specific gene silencing technology triggered by small interfering RNA (siRNA), represents promising new avenues for treatment of various liver diseases including hepatitis C virus (HCV) infection. In plants and invertebrates, RNAi provides an important mechanism of cellular defence against viral pathogens and is dependent on the spread of siRNA to neighbouring cells. A study was undertaken to investigate whether vector-delivered RNAi can transfer between hepatic cells in vitro and in mice, and whether this exchange could extend the therapeutic effect of RNAi against HCV infection. Methods Transmission of RNAi was investigated in culture by assessing silencing of HCV replication and expression of viral entry receptor CD81 using a human hepatic cell line and primary B lymphocytes transduced with siRNA-expressing vectors. In vivo transmission between hepatic cells was investigated in NOD/SCID mice. Involvement of exosomes was demonstrated by purification, uptake and mass spectrometric analysis. Results Human and mouse liver cells, as well as primary human B cells, were found to have the ability to exchange small RNAs, including cellular endogenous microRNA and delivered siRNA targeting HCV or CD81. The transmission of RNAi was largely independent of cell contact and partially mediated by exosomes. Evidence of RNAi transmission in vivo was observed in NOD/SCID mice engrafted with human hepatoma cells producing CD81 siRNA, causing suppression of CD81 expression in mouse hepatocytes. Conclusion Both human and mouse hepatic cells exchange small silencing RNAs, partially mediated by shuttling of exosomes. Transmission of siRNA potentially extends the therapeutic reach of RNAi-based therapies against HCV as well as other liver diseases.

Activated tumor‐infiltrating CD4+ regulatory T cells restrain antitumor immunity in patients with primary or metastatic liver cancer

The mechanisms that enable liver cancer to escape elimination by the immune system remain unclear, but their elucidation may provide novel therapeutic interventions. We investigated the influence of tumor‐infiltrating regulatory T cells on tumor‐specific T cell responses in patients with liver cancer, using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC) or liver metastases from colorectal cancer (LM‐CRC). Here we report that in both HCC and LM‐CRC, CD4+CD25+Foxp3+ regulatory T cells (Tregs) accumulate in the tumor milieu and are potent suppressors of autologous tumor‐specific T cell responses. Especially in LM‐CRC, where Treg accumulation is more prominent, there is good evidence for local proliferation of Tregs at the cancer site. We show that tumor Tregs up‐regulate the expression of glucocorticoid‐induced tumor necrosis factor receptor (GITR) compared with Tregs in tumor‐free liver tissue and blood. Importantly, treatment with soluble GITR ligand (GITRL) induces a decrease in the suppression mediated by the activated tumor‐infiltrating Tregs and restores the proliferative capacity and cytokine production of CD4+CD25− T cells. Conclusion: Our results show that tumor‐associated Tregs are critical for immune evasion in liver cancer, and we propose that GITRL constitutes a rational treatment for this disease. (HEPATOLOGY 2013)

Barrett's oesophagus is characterized by a predominantly humoral inflammatory response

Barretts oesophagus (BO) is thought to be an intermediate step in the progression from reflux oesophagitis (RO) to oesophageal adenocarcinoma. Premalignant conditions that develop in the presence of chronic inflammation are often associated with the development of a more pronounced humoral immune response during progression of the disease. The aim of this study was to determine whether BO is also associated with a more pronounced humoral immune response when compared to RO. Immunohistochemical studies were performed to quantify the mean numbers of Th2 effector cells (plasma cells and mast cells) and Th1 effector cells (macrophages and CD8+ T cells) to detect the antibody classes produced by plasma cells (IgA, IgG, IgM or IgE) and to determine the presence of isolated lymph follicles [segregated B and T cell areas, follicular dendritic cells (CD23) and expression of CXCL13] in 124 oesophageal biopsies from 20 patients with BO and 20 patients with RO. The proportion of Th2 effector cells was higher in BO than in RO, mainly due to higher numbers of plasma cells and mast cells in BO (p < 0.001). Most plasma cells in BO and RO expressed IgG, but several IgE+ plasma cells were detected in BO: these were rare in RO. In line with this, isolated lymph follicles were observed in 4/20 (20%) patients with BO, but not in RO. We therefore conclude that the inflammatory response is skewed towards a more pronounced humoral immune response when RO progresses to BO. It may be that this shift, which is similar to that found in other chronic inflammatory conditions, contributes to an increased cancer risk in BO. Copyright

Conversion from calcineurin inhibitor to mycophenolate mofetil-based immunosuppression changes the frequency and phenotype of CD4+FOXP3+ regulatory T cells.

Background. CD4+Foxp3+ regulatory T cells (Treg) depend on interleukin (IL)-2 for their function and survival. By interfering with the IL-2 production, calcineurin inhibitors (CNI) may negatively affect Treg. Here, we describe the effects of conversion from CNI to mycophenolate mofetil (MMF) monotherapy on renal function, and on Treg frequency and phenotype in liver transplant recipients. Methods. Patients (n=16) with renal impairment on CNI were converted to MMF and received a single dose of IL-2-receptor blocking antibody (Daclizumab). Control patients (n=8) continued CNI treatment. Results. Renal function rapidly and significantly improved after conversion. Daclizumab treatment resulted in a 75% blocking of CD25 at 1 month causing a significant reduction in the percentage of CD4+CD25+ cells but not affecting the percentage of CD4+CD25+Foxp3+ cells. Six months after conversion to MMF, the percentage of CD4+CD25+Foxp3+ cells increased significantly by 125%. FOXP3 mRNA analysis of mononuclear cells confirmed the enrichment of Foxp3 in peripheral blood. Interestingly, the CD25 expression level on CD4+Foxp3+, but not CD4+Foxp3−, cells significantly increased compared with preconversion. Conclusion. Conversion to MMF increases the percentage and CD25 expression of CD4+Foxp3+ cells indicating that MMF therapy can overturn the repressive effect of CNI on circulating Treg levels and therefore may promote Treg-mediated suppression of alloreactivity.

Characterization of human liver dendritic cells in liver grafts and perfusates.

It is generally accepted that donor myeloid dendritic cells (MDC) are the main instigators of acute rejection after organ transplantation. The aim of the present study was to characterize MDC in human donor livers using liver grafts and perfusates as a source. Perfusates were collected during ex vivo vascular perfusion of liver grafts pretransplantation. MDC, visualized in wedge biopsies by immunohistochemistry with anti‐BDCA‐1 monoclonal antibody (mAb), were predominantly observed in the portal fields. Liver MDC, isolated from liver wedge biopsies, had an immature phenotype with a low expression of CD80 and CD83. Perfusates were collected from 20 grafts; perfusate mononuclear cells (MNC) contained 1.5% (range, 0.3‐6.6%) MDC with a viability of 97 ± 2%. Perfusates were a rich source of hepatic MDC since 0.9 × 106 (range, 0.11‐4.5 × 106) MDC detached from donor livers during vascular perfusion pretransplantation. Perfusate MDC were used to further characterize hepatic MDC. Perfusate MDC expressed less DC‐LAMP (P = 0.000), CD80 (P = 0.000), CD86 (P = 0.003), and CCR7 (P = 0.014) than mature hepatic lymph node (LN) MDC, and similar CD86 (P = 0.140) and CCR7 (P = 0.262) as and more DC‐LAMP (P = 0.007) and CD80 (P = 0.002) than immature blood MDC. Perfusate MDC differed from blood MDC in producing significantly higher amounts of interleukin (IL)‐10 in response to lipopolysaccharide (LPS), and in being able to stimulate allogeneic T‐cell proliferation. In conclusion, human donor livers contain exclusively immature MDC that detach in high numbers from the liver graft during pretransplantation perfusion. These viable MDC have the capacity to stimulate allogeneic T‐cells, and thus may represent a major player in the induction of acute rejection. Liver Transpl 12: 384–393, 2006.