Haiming Wei

Invariant NKT cells promote alcohol-induced steatohepatitis through interleukin-1β in mice

BACKGROUND & AIMSnIt was reported that alcohol consumption activated the NLRP3 inflammasome in Kupffer cells, leading to mature interleukin (IL)-1β release in alcoholic liver injury; however, how IL-1β promotes liver injury remains unclear.nnnMETHODSnWe investigated the role of IL-1β in alcoholic steatohepatitis by using a chronic plus single-binge ethanol consumption mouse model.nnnRESULTSnHere, liver steatosis was accompanied by notably increased invariant natural killer T (iNKT) cell numbers and activation, and iNKT-deficient Jα18(-/-) mice developed less alcohol-induced steatosis, with reduced liver inflammation and neutrophil infiltration. Kupffer cells and IL-1β were required for the hepatic iNKT accumulation, as either blocking IL-1β signaling with a recombinant IL-1 receptor antagonist (IL-1Ra), depleting Kupffer cells by clodronate liposomes, or specifically silencing IL-1β in Kupffer cells by nanoparticle-encapsulated siRNA, resulted in inhibited hepatic iNKT cell accumulation and activation, as well as amelioration of alcoholic fatty liver. In addition, IL-1β overexpression in hepatocytes was sufficient to compensate for Kupffer cell depletion. Increased gene and protein expression of mature IL-1β correlated with elevated expression of the NLRP3 inflammasome components NLRP3, ASC, and cleaved caspase-1 in Kupffer cells from ethanol-exposed wild-type mice. NLRP3 deficiency led to the attenuation of alcoholic steatosis, similarly as Kupffer cell depletion, almost without hepatic NKT cells.nnnCONCLUSIONSnAfter alcohol-exposure Kupffer cell-derived IL-1β triggered by NLRP3 activation, recruits and activates hepatic iNKT cells, subsequently promoting liver inflammation and neutrophil infiltration, and inducing alcoholic liver injury.

Tumor-released Galectin-3, a Soluble Inhibitory Ligand of Human NKp30, Plays an Important Role in Tumor Escape from NK Cell Attack

Background: Galectin-3, a β-galactoside-binding protein, is expressed by many types of tumor cells. Results: Galectin-3 secreted from a tumor works as a soluble inhibitory ligand of the NK cell receptor NKp30 to inhibit NK cell immune responses against tumors. Conclusion: Galectin-3 regulates the antitumor immunity of human NK cells. Significance: This novel mechanism may provide a new therapeutic target for tumor treatment. Human Galectin-3 (Gal-3), a β-galactoside-binding protein expressed by tumor cells, has been reported to act as an immune regulator in antitumor T cells. However, its effect on natural killer (NK) cells is elusive. Using a recombinant human NK cell-activating receptor, NKp30 fusion protein (NKp30-Fc), we found that soluble NKp30-Fc could immunoprecipitate Galectin-3. The direct interaction between NKp30 and Galectin-3 was further confirmed using surface plasmon resonance experiments. Because Galectin-3 was mainly released from tumor cells in a soluble form in our study, the binding assay was performed to show that soluble Galectin-3 specifically bound to NK cells and NKp30 on the surface of the NK cells. Functionally, when soluble Galectin-3 was added to the NK-tumor cell coculture system, the NKp30-mediated, but not NKG2D-mediated, cytolysis and CD107a expression in the NK cells were inhibited, and these phenotypes could be restored by preincubation of soluble Galectin-3 with NKp30-Fc fusion protein or the addition of anti-Gal-3 antibody alone. Moreover, genetic down-regulation of Galectin-3 (shGal-3) resulted in tumor cells being more sensitive to NK cell lysis, and, reversely, Galectin-3-overexpressing HeLa cells (exGal-3) became less sensitive to NK cell killing. The results of these in vitro experiments were supported by studies in shGal-3-HeLa or exGal-3-HeLa xenograft non-obese diabetic/severe combined immunodeficiency mice after NK cell adoptive immunotherapy, indicating that Galectin-3 strongly antagonizes human NK cell attack against tumors in vivo. These findings indicate that Galectin-3 may function as an immune regulator to inhibit NK cell function against tumors, therefore providing a new therapeutic target for tumor treatment.

T‐cell Ig and ITIM domain regulates natural killer cell activation in murine acute viral hepatitis

Uncontrolled natural killer (NK) cell activation during the early response to acute viral infection can lead to severe immunopathology, and the mechanisms NK cells use to achieve self‐tolerance in such contexts are currently unclear. Here, NK cells up‐regulated a coinhibitory receptor, T‐cell Ig and ITIM domain (TIGIT), during challenge with the viral double‐stranded RNA (dsRNA) analog poly I:C. Blocking TIGIT by antibody treatment in vivo or a genetic deficiency in Tigit enhanced NK cell activation and aggravated liver injury in a poly I:C/D‐GalN‐induced model of acute fulminant hepatitis, suggesting that TIGIT is normally required for protecting against NK cell‐mediated liver injury. Furthermore, adoptively transferring Tigit−/− NK cells into NK cell‐deficient Nfil3−/− mice also resulted in elevated liver injury. Reconstituting Kupffer cell‐depleted mice with poliovirus receptor (PVR/CD155, a TIGIT ligand)‐silenced Kupffer cells led to aggravated liver injury in a TIGIT‐dependent manner. Blocking TIGIT in an NK‐Kupffer cell coculture in vitro enhanced NK cell activation and interferon‐gamma (IFN‐γ) production in a PVR‐dependent manner. We also found that TIGIT was up‐regulated selectively on NK cells and protected against liver injury in an acute adenovirus infection model in both an NK cell‐ and Kupffer cell‐dependent manner. Knocking down PVR in Kupffer cells resulted in aggravated liver injury in response to adenovirus infection in a TIGIT‐dependent manner. Conclusion: TIGIT negatively regulates NK‐Kupffer cell crosstalk and alleviates liver injury in response to poly I:C/D‐GalN challenge or acute adenovirus infection, suggesting a novel mechanism of NK cell self‐tolerance in liver homeostasis during acute viral infection. (Hepatology 2014;59:1715–1725)

TIGIT safeguards liver regeneration through regulating natural killer cell‐hepatocyte crosstalk

Overactivation of innate immunity, particularly natural killer (NK) cells, is harmful to liver regeneration; however, the molecular mechanisms that limit NK cell overactivation during liver regeneration are still elusive. Here we show that a coinhibitory receptor, T cell Ig and ITIM domain (TIGIT), was selectively up‐regulated on NK cells, along with high expression of its ligand, poliovirus receptor (PVR/CD155), on hepatocytes during liver regeneration. The absence of TIGIT impaired liver regeneration in vivo, along with overactivation of NK cells and higher NK‐derived interferon‐gamma (IFN‐γ) production. We also show that both depletion of NK cells and deficiency of IFN‐γ, but not deficiency of RAG1, rescued impaired liver regeneration caused by the absence of TIGIT. Adoptive transfer of Tigit–/– NK cells into NK‐deficient Nfil3–/– mice sufficiently led to impairment of liver regeneration. On the other hand, silencing PVR in hepatocytes rescued impaired liver regeneration caused by TIGIT deficiency in vivo, while blockade of TIGIT in NK‐hepatocyte coculture increased IFN‐γ production by NK cells in vitro. Conclusion: TIGIT is a safeguard molecule to improve liver regeneration through negatively regulating NK‐hepatocyte crosstalk. This finding suggests a novel mechanism of NK cell self‐tolerance towards regenerative hyperplasia of the host. (Hepatology 2014;60:1389–1398)

Recombinant soluble CD226 protein directly inhibits cancer cell proliferation in vitro

Interactions between CD155 and nectins on tumor cells have been reported to potentially inhibit tumor growth. CD226, a receptor that recognizes CD155 and CD112, is an activation receptor of NK and T cells by which immune cells may attack a tumor. The purpose of this study is to explore whether soluble CD226 (sCD226) directly inhibits tumor growth by binding CD155 or CD112 on tumor cells. We expressed, purified and confirmed the identity of recombinant sCD226 (19aa-248aa) and then examined the effect of sCD226 on tumor cell growth using CD226 ligand (CD155 and CD112)-expressing cancer cell lines (K562, HeLa). After 3days of co-culture with sCD226, we found that the numbers of K562 and HeLa cells were significantly reduced but those of a CD226-blocking mAb specifically attenuated the inhibitory effects of sCD226. We also noted that the sCD226 protein could compete with a PE-conjugated anti-CD112 antibody in flow cytometric analysis and block the binding of the PE-conjugated anti-CD112 antibody to tumor cells. Mechanistic studies using flow cytometric analysis demonstrated that sCD226 inhibited the division of CFSE (carboxyfluorescein diacetate succinimidyl ester)-labeled K562 cells by delaying the cell cycle. In addition, we observed that sCD226 might have an impact on the metastatic potential of solid tumors in vitro. These results demonstrated that sCD226 molecule might be a potential biotherapy against tumor for further development.

CD226 Protein Is Involved in Immune Synapse Formation and Triggers Natural Killer (NK) Cell Activation via Its First Extracellular Domain

Background: CD226 is an activating receptor on NK cells that mediates NK cell cytotoxicity. Results: The first extracellular domain of CD226 (CD226-ECD1) mediates NK cell recognition, adhesion, immune synapse formation, and cytotoxicity against target cells. Conclusion: CD226-ECD1 retains almost all functions of the full-length CD226 protein. Significance: The conclusion is helpful to understand the mechanism by which CD226 recognizes its ligands. CD226, an activating receptor that interacts with the ligands CD155 and CD112, activates natural killer (NK) cells via its immunoreceptor tyrosine-based activatory motif (ITAM). There are two extracellular domains of CD226; however, the comparative functional relevance of these domains remains unknown. In this study, two different deletion mutants, rCD226-ECD1 (the first extracellular domain) and rCD226-ECD (full extracellular domains), were recombinantly expressed. We observed that rCD226-ECD1, similar to rCD226-ECD, specifically bound to ligand-positive cell lines and that this interaction could be competitively blocked by an anti-CD226 mAb. In addition, rCD226-ECD1 was able to block the binding of CD112 mAb to tumor cells in a competitive binding assay. Importantly, based on surface plasmon resonance (SPR), we determined that rCD226-ECD1, similar to rCD226-ECD, directly bound to its ligand CD155 on a protein chip. Functionally, NK cell cytotoxicity against K562 or HeLa cells was blocked by rCD226-ECD1 by reducing the expression of CD69 and granzyme B, indicating the critical role of ECD1 in NK cell activation. We also examined the role of rCD226-ECD1 in effector/target interactions by using rCD226-ECD to block these interactions. Using flow cytometry, we found that the number of conjugates between IL-2-dependent NKL cells and HeLa cells was reduced and observed that the formation of immune synapses was also decreased under confocal microscopy. In addition, we prepared two anti-rCD226-ECD1 agonistic antibodies, 2E6 and 3B9. Both 2E6 and 3B9 antibodies could induce the phosphorylation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.

CRACC-CRACC Interaction between Kupffer and NK Cells Contributes to Poly I:C/D-GalN Induced Hepatitis

CD2-like receptor activating cytotoxic cells (CRACC) is known as a critical activating receptor of natural killer (NK) cells. We have previously reported that NK cells contribute to Poly I:C/D-galactosamine (D-GalN)-induced fulminant hepatitis. Since natural killer group 2, member D (NKG2D) is considered critical but not the only activating receptor for NK cells, we investigated the role of CRACC in this model. We found that CRACC was abundant on hepatic NK cells but with low expression levels on Kupffer cells under normal conditions. Expression of CRACC on NK cells and Kupffer cells was remarkably upregulated after poly I:C injection. Hepatic CRACC mRNA levels were also upregulated in Poly I:C/D-GalN-treated mice, and correlated positively with the serum alanine aminotransferase (ALT) levels. CRACC expression on Kupffer cells was specifically silenced by nano-particle encapsulated siRNA in vivo, which significantly reduced Poly I:C/D-GalN-induced liver injury. In co-culture experiments, it was further verified that silencing CRACC expression or blockade of CRACC activation by mAb reduced the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Collectively, our findings suggest that CRACC-CRACC interaction between NK cells and resident Kupffer cells contributes to Poly I:C/D-GalN-induced fulminant hepatitis.

Characterization of human B cells in umbilical cord blood-transplanted NOD/SCID mice

Humanized mice are crucially important for preclinical studies. However, the development and potential function of human B cells in chimeras remain unclear. Here, we describe the study of human B cells in NOD/LtSzPrkdcscid/J (NOD/SCID) mice. In this study, we transplanted 1.0×10(5) human CD34(+) cells from umbilical cord blood (UCB) into NOD/SCID mice after pretreatment with anti-asialo GM1 antiserum and sublethal irradiation. Human CD45(+) cells were detected in the peripheral blood of the recipient mice from 6 weeks after transplantation. CD19(+) B cells accounted for the greater part of the CD45(+) cells in the human UCB-chimeric mice, but their maturational stages differed in different organs. Most of the bone marrow (BM) CD19(+) cells were immature IgM(-)IgD(-)CD24(hi)CD38(hi) B cells, whereas the mature CD5(+)IgM(+)IgD(+)CD24(int)CD38(int)CD19(+) B cells were predominantly present in the spleen and peripheral blood. Human immunoglobulin (Ig) M was detected in mouse plasma. The human B cells also secreted human interleukin-10 after stimulation with LPS in vitro. These results show that human CD34(+) cells can differentiate into human B cells in NOD/SCID mice, with development and functions that are similar to those of B cell subsets in humans. The transplantation of human CD34(+) cells into NOD/SCID mice may provide a useful tool to study the development and function of human B cells.

Efficient attenuation of NK cell-mediated liver injury through genetically manipulating multiple immunogenes by using a liver-directed vector.

Adenovirus or adenoviral vectors were reported to induce serious liver inflammation in an NK cell–dependent manner, which limits its clinical applicability for liver gene therapy. We tried to develop an efficient liver-directed therapeutic approach to control hepatic NK cell function via simultaneously manipulating multiple immune genes. Based on our previous study, we found that CCL5 knockdown synergistically enhanced the attenuating effect of silencing CX3CL1 (fractalkine [FKN]) in adenovirus-induced acute liver injury. In addition, the combined treatment of human IL-10 expression with FKN knockdown would further strengthen the protective effect of silencing FKN. We used a hepatocyte-specific promoter to construct a hepatocyte-specific multiple function vector, which could simultaneously overexpress human IL-10 and knock down CCL5 and FKN expression. This vector could attenuate adenovirus-induced acute hepatitis highly efficiently by reducing liver NK cell recruitment and serum IFN-γ and TNF-α. The multiple function vectors could be delivered by nonviral (hydrodynamic injection) and viral (adenovirus) approaches, and maintained long-term function (more than 1 month in mice). Our results suggest a possible strategy to ameliorate the acute liver injury induced by adenovirus by modulating multiple immune genes. The novel multifunction vector has an extensive and practical use for polygenic and complex liver diseases such as malignancies and hepatitis, which correlate with multiple gene disorders.

Preparation and functional identification of a monoclonal antibody against the recombinant soluble human NKp30 receptor.

NKp30 is an important activating receptor of human natural killer (NK) cells that participates in NK cell activation and cytotoxicity against tumor and infected cells. To study the function of NKp30, anti-human NKp30 monoclonal antibody was prepared. The human NKp30 ectodomain (rhNKp30) was expressed in Escherichia coli as inclusion bodies and refolded using the dilution method. The refolded rhNKp30 was purified by immobilized metal affinity chromatography. The activity of soluble rhNKp30 was confirmed by flow cytometry and NK cytotoxicity assays. Four hybridoma cell lines producing monoclonal antibodies against rhNKp30 were obtained. One of the monoclonal antibodies, designated as 3G5, was highly specific and could be used in western blotting, immunoprecipitation, ELISA, and flow cytometry assays. The preparation of soluble rhNKp30 and a monoclonal antibody against NKp30 may provide useful tools for further functional studies of human NKp30.