A. Castellaneta

NOD2 Ligation Subverts IFN-α Production by Liver Plasmacytoid Dendritic Cells and Inhibits Their T Cell Allostimulatory Activity via B7-H1 Up-Regulation

The nucleotide-binding oligomerization domain (NOD)2/CARD15 protein, which senses muramyl dipeptide (MDP), a product of bacterial peptidoglycan, appears to play an important role in regulating intestinal immunity. Although the liver is exposed to gut-derived MDP, the influence of NOD2 ligation on hepatic APC, in particular dendritic cells (DC), is unknown. Freshly isolated mouse liver and spleen plasmacytoid (p)DC expressed higher levels of NOD2 message than conventional myeloid (m)DC. Following MDP stimulation in vivo, liver pDC, but not mDC, up-regulated expression of IFN regulatory factor 4 (IRF-4), a negative regulator of TLR signaling, and induced less allogeneic T cell proliferation and IFN-γ production. The adoptive transfer of liver pDC from MDP-treated mice failed to prime allogeneic T cells in vivo. By contrast, splenic DC IRF-4 levels and T cell stimulatory activity remained unchanged. Liver pDC from MDP-stimulated mice also displayed greater IκBα, cell surface B7-H1, and B7-H1 relative to CD86 than control liver pDC. No similar effects were observed for liver mDC or spleen DC. Absence of B7-H1 on liver pDC reversed the inhibitory effect of MDP. After ex vivo stimulation with LPS or CpG, liver pDC but not mDC from MDP-treated animals secreted less IL-12p70, IL-6, and TNF-α and induced weaker allogeneic T cell proliferation than those from controls. Moreover, CpG-stimulated liver pDC from MDP-treated mice secreted less IFN-α than their splenic counterparts, and systemic levels of IFN-α were reduced in MDP-treated animals after CpG administration. These findings suggest that differential effects of NOD2 ligation on liver pDC may play a role in regulating hepatic innate and adaptive immunity.

Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo.

Chromatin‐modifying HDACi exhibit anti‐inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM‐CSF‐stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b+Ly6C+F4/80intCD115+), which suppressed allogeneic T cell proliferation in a NOS‐ and HO‐1‐dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin–Sca‐1+c‐Kit+). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM‐CSF administration and increased the number of CD11b+Gr1+ cells in BM and spleen. Increased numbers of Gr1+ cells, which suppressed T cell proliferation, were recovered from spleens of TSA‐treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti‐inflammatory action of HDACi.

Monitoring the operationally tolerant liver allograft recipient.

Purpose of reviewA fundamental goal in transplantation is the establishment of allograft function without ongoing immunosuppression. Robust allograft tolerance has been established in experimental transplantation models, whereas clinical operational tolerance has been described most frequently following human liver transplantation. Recent findingsClinical assessment of tolerance has been limited to laboratory evaluation of organ function. Additional tools include graft monitoring through biopsy and blood sampling for biomarker analysis. Current biomarkers under assessment in recent years include dendritic cell subsets, regulatory T cells, antidonor antibodies, and gene polymorphisms. Emerging microarray analysis that is being prospectively validated will also be reviewed.A further tool in the characterization of the tolerant patient will be the accurate enrollment of such patients into a multicenter registry that will prospectively follow the natural history of the patient withdrawn from immunosuppression and help facilitate the entry of interested patients to mechanistic and immune monitoring trials. The International Solid Organ Transplant Tolerance Registry (www.transplant-tolerance.org) will be briefly described. SummaryEffective biomarker characterization of the operationally tolerant liver allograft recipient would allow earlier, well tolerated, prospective drug withdrawal with the goal of extending the potential benefits of drug minimization to an increasing number of patients in a more predictable fashion.

Plasmacytoid dendritic cell‐derived IFN‐α promotes murine liver ischemia/reperfusion injury by induction of hepatocyte IRF‐1

Plasmacytoid dendritic cells (pDC) constitute the bodys 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. (Hepatology 2014;60:267–277)

Functional modification of CD11c+ liver dendritic cells during liver regeneration after partial hepatectomy in mice†

Local immunosuppression within the liver and sex steroid changes, in both blood and tissue during liver regeneration, are well‐recognized events. Dendritic cells (DC) play pivotal roles in the induction and regulation of immune responses. Their numbers are expanded markedly in vivo by fms‐like tyrosine kinase 3 ligand (Flt3L) administration, without modification of their maturation state. Recent evidence suggests that estrogen can modulate DC function and promote a Th2‐type immune response. Few data are available concerning the role of DC in liver regeneration. After 75% partial hepatectomy (PH) in male C57BL/6 mice, CD11c+ liver (L)DC increased significantly within 6 hours and maintained an immature phenotype. Numbers returned to pre‐hepatectomy levels by 24 hours. The expanded LDC population showed increased IL‐10 and reduced IFN‐γ gene transcription. Using these DC compared with control LDC as T cell stimulators in 72‐hour mixed leukocyte cultures, IL‐10 production was enhanced and IFN‐γ production reduced. LDC isolated 6 hours after 75% PH exhibited enhanced estrogen receptor (ER) expression, concomitant with increased serum estrogen levels. By contrast, spleen (S)DC isolated before and after PH showed no significant changes in their function (maturation state, T cell stimulatory activity, cytokine production, and ER expression). Increased liver regeneration (more than 50%) was observed 48 hours after 40% PH in the Flt3L‐pretreated compared with the PBS group. In conclusion, interstitial LDC may play a key role in local immune regulation during liver regeneration, possibly linking estrogen‐mediated immune modulation and hepatocyte proliferation. (HEPATOLOGY 2006;43:807–816.)

Heart, but not skin, allografts from donors lacking Flt3 ligand exhibit markedly prolonged survival time.

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L−/−) show severe reductions in both myeloid (CD11c+CD8α−) and lymphoid-related DC (CD11c+CD8α+) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c+ interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c+ cells that migrated from Flt3L−/− skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c+ cells. We examined the survival of Flt3L−/− heart or tail skin grafts (H2b) in allogeneic wt (BALB/c; H2d) recipients. The outcome of transplantation of BALB/c organs into Flt3L−/− recipients was also determined. Flt3L−/− mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L−/− heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L−/− hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L−/− hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L−/− recipients reflects presumably an intact role of the direct pathway of allorecognition.

Mycophenolate mofetil in the treatment of autoimmune HCV‐associated haematological disorders showing steroid resistance or dependence

Summary. We report two cases of hepatitis C virus (HCV) associated autoimmune haematological disorders successfully treated with an unusual protocol (mycophenolate mofetil: MMF). The first case was a male patient with chronic HCV infection who developed, during interferon (IFN)/ribavirin therapy, severe autoimmune thrombocytopenia unresponsive to steroids. MMF was then administered and, simultaneously, the steroid dose was gradually reduced until withdrawal. Following this strategy, a progressive increase in platelet count and complete negativity of anti‐PLT antibodies were achieved without changes in HCV‐RNA quantitative determination. The second case was a woman with HCV liver cirrhosis with severe anaemia and Coombs test positivity partially responsive to continuous administration of steroid high doses. However, this treatment unmasked a severely painful vertebral osteoporosis. For this reason we introduced MMF and simultaneously steroid therapy was progressively reduced until withdrawal. Haemoglobin reached a normal value and the Coombs test became negative within 60 days. These case reports suggest that MMF may represent an interesting therapeutic approach for autoimmune HCV associated haematological disorders.

Antiviral activity and safety profile of silibinin in HCV patients with advanced fibrosis after liver transplantation: a randomized clinical trial.

Response to interferon‐based therapies in HCV recurrence after liver transplantation (LT) is unsatisfactory, and major safety issues aroused in preliminary experience with boceprevir and telaprevir. As transplant community identified HCV viral clearance as a critical matter, efficacious and safe anti‐HCV therapies are awaited. The aim of this study was to assess efficacy and safety of intravenous silibinin monotherapy in patients with established HCV recurrence after LT, nonresponders to pegylated interferon and ribavirin. This is a single center, prospective, randomized, parallel‐group, double‐blind, placebo‐controlled, phase 2 trial including 20 patients randomly assigned (3:1) to receive daily 20 mg/kg of intravenous silibinin or saline as placebo, for 14 consecutive days. On day 14 of treatment, viral load decreased by 2.30 ± 1.32 in silibinin group versus no change in the placebo group (P = 0.0002). Sixteen days after the end of the treatment, viral load mean values were similar to baseline. Treatment resulted well tolerated apart from a transient and reversible increase in bilirubin. Neither changes in immunosuppressant through levels nor dosage adjustments were necessary. Silibinin monotherapy has a significant antiviral activity in patients with established HCV recurrence on the graft not responding to standard therapy and confirms safety and tolerability without interaction with immunosuppressive drugs (ClinicalTrials.gov number: NCT01518933).

Effect of gonadectomy on HCC development in HBV transgenic mice.

BACKGROUND AND AIMS Epidemiological data demonstrate that HCC is prevalent in men compared to women. Herein, we examined the effect of gonadectomy in a murine model that spontaneously develops HCC. ANIMALS AND METHODS Thirty-two male and 26 female HBV transgenic mice [Tg (Alb-1 HBV) Bri 44] underwent surgical castration or sham operation. At the 18th month, serum samples were collected and all mice were sacrificed. Liver weight and volume were evaluated, each liver was cut into 1.5-mm-thick consecutive slices and nodules were examined on freshly isolated tissue. Consecutive histological sections obtained from each liver slice were evaluated to confirm the diagnosis of HCC. RESULTS Sham-operated females showed a significantly lower neoplastic growth compared to sham-operated males. This difference disappeared when females underwent gonadectomy. In males, neoplastic growth was not influenced by gonadectomy. Testosterone and estradiol levels were profoundly modified by gonadectomy in both males and females. The testosterone/estradiol ratio in gonadectomized females increased 4.5-fold compared to that in sham-operated females, becoming more similar to the ratio observed in castrated and sham-operated male mice. CONCLUSIONS HCC growth in our experimental model was not simply influenced by the levels of testosterone or estradiol, taken singularly, but depended on their ratio.

Possible involvement of androgen receptor alterations in hepatocarcinogenesis

BACKGROUND Androgen receptors (ARs) act as transcription factors. An increased AR activity could be due either to mutations or to an increased expression of the receptor. AR mutations involving the hormone binding domain could increase AR function and promote carcinogenesis, as suggested for prostate cancer. AIMS Herein, we evaluated qualitative (point mutations involving the hormone binding domain) and quantitative AR alterations and their possible correlation with cell proliferation and tumour grading. MATERIALS Carcinomatous and non-cancerous surrounding liver tissue was collected from 14 Caucasian patients with hepatocarcinoma. They were all affected by cirrhosis with different aetiologies. METHODS AR missense mutations, AR mRNA and protein levels, AR distribution in the liver, liver cell proliferation, and tumour staging were evaluated by DNA sequencing, quantitative real-time PCR, Western blot analysis, immunofluorescence, PCNA immunostaining, and conventional histological techniques, respectively. RESULTS AR gene regions encoding the hormone binding domain did not contain any missense mutation. AR mRNA and protein levels were increased in hepatocarcinoma compared to non-cancerous surrounding tissue. Cell proliferation was significantly increased in the tumour compared to non-cancerous surrounding tissue. CONCLUSIONS Mutations of the AR regions studied were not involved in hepatocarcinogenesis. Elevated AR levels in transformed cells could have a tumour promoting effect by stimulating cell growth.