Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice

Hepatitis B virus (HBV) infection is one of the most frequent causes of chronic liver disease worldwide. Because recent studies have suggested that Toll-like receptor (TLR)-based therapies may be a promising approach in the treatment of HBV infection, we studied the role of the local innate immune system of the liver as a possible mediator of this effect. Murine nonparenchymal cells, including Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs), were isolated from C57/BL6 wild-type or MyD88(-/-) mice and stimulated by agonists of TLR1 to TLR9. Supernatants were harvested and assayed for their antiviral activity against HBV in HBV-Met cells. No direct antiviral effect of TLR agonists could be observed. In controls (myeloid dendritic cells), TLR1, TLR3, TLR4, TLR7, and TLR9 activation lead to production of antiviral cytokines. By contrast, only supernatants from TLR3-stimulated and TLR4-stimulated KCs and TLR3-stimulated LSECs from wild-type mice were able to potently suppress HBV replication as assessed via Southern blotting. Similar results were found with cells from MyD88(-/-) mice, indicating that the effect was independent of this signaling pathway. Cellular HBV RNA and hepatitis B surface antigen or hepatitis B e antigen levels in supernatants remained unchanged. Using neutralizing antibodies, we demonstrated that the TLR3-mediated effect but not the TLR4-mediated effect is mediated exclusively through interferon-beta. Conclusion: Our data indicate that the innate immune system of the liver can control HBV replication after activation by TLR agonists. This has implications for the development of TLR-based therapeutic approaches against HBV.     

Toll-like receptor-initiated testicular innate immune responses in mouse Leydig cells

The testis is an immunoprivileged site, where the local cell-initiated testicular innate immune responses play a crucial role in defense against microbial infections. Mechanisms modulating the testicular cell-built defense system remain to be clarified. In this article, we demonstrate that Leydig cells, a major cell population in the testicular interstitium, initiate innate immunity through the activation of Toll-like receptors (TLRs). Several TLRs are expressed in mouse Leydig cells; among these, TLR3 and TLR4 are expressed at relatively high levels compared with other TLR members. Both TLR3 and TLR4 can be activated by their agonists (polyinosinic:polycytidylic acid and lipopolysaccharide) in Leydig cells and subsequently induce the production of inflammatory factors, such as IL-1β, IL-6, TNF-α, and type 1 interferons (IFN) (IFN-α and IFN-β). Notably, the activation of TLR3 and TLR4 suppresses steroidogenesis by Leydig cells. Further, we provide evidence that Axl and Mer receptor tyrosine kinases are expressed in Leydig cells and regulate TLR-mediated innate immune responses negatively. Data presented here describe a novel function of Leydig cells in eliciting testicular innate immune responses that should contribute to the protection of the testis from microbial infections.     

Distinct single-component adjuvants steer human DC-mediated T-cell polarization via Toll-like receptor signaling toward a potent antiviral immune response

The COVID-19 pandemic highlights the importance of efficient and safe vaccine development. Vaccine adjuvants are essential to boost and tailor the immune response to the corresponding pathogen. To allow for an educated selection, we assessed the effect of different adjuvants on human monocyte-derived dendritic cells (DCs) and their ability to polarize innate and adaptive immune responses. In contrast to commonly used adjuvants, such as aluminum hydroxide, Toll-like receptor (TLR) agonists induced robust phenotypic and functional DC maturation. In a DC-lymphocyte coculture system, we investigated the ensuing immune reactions. While monophosphoryl lipid A synthetic, a TLR4 ligand, induced checkpoint inhibitors indicative for immune exhaustion, the TLR7/8 agonist Resiquimod (R848) induced prominent type-1 interferon and interleukin 6 responses and robust CTL, B-cell, and NK-cell proliferation, which is particularly suited for antiviral immune responses. The recently licensed COVID-19 vaccines, BNT162b and mRNA-1273, are both based on single-stranded RNA. Indeed, we could confirm that the cytokine profile induced by lipid-complexed RNA was almost identical to the pattern induced by R848. Although this awaits further investigation, our results suggest that their efficacy involves the highly efficient antiviral response pattern stimulated by the RNAs’ TLR7/8 activation.

The current COVID-19 pandemic has unprecedently spurred vaccine development and has so far resulted in over 287 new vaccine candidates under evaluation (1–3).In contrast to the vaccine formulations with inactivated or live-attenuated whole pathogens, the use of only purified antigens of the pathogen in today’s subunit vaccines is well tolerated and deemed to be safe. These antigens provide high specificity, but their low intrinsic immunogenicity requires the combined application with an adjuvant to activate the immune system (4). Adjuvants are not unified by structure, target, or mechanism of action (MoA), but they all shape, direct, and potentiate the immune response. The selection of an appropriate adjuvant is essential for vaccine efficacy (5).Aluminum hydroxide [Al(OH)₃] and MF59 belong to the first generation of adjuvants, but even though Al(OH)₃ has a good safety profile, its MoA is still not fully understood (5, 6). Its limitation of stimulating a strong humoral response (7) has driven the development of second-generation adjuvants such as pattern recognition receptor (PRR) ligands or plant-derived compounds (e.g., saponins) to induce specific cell-mediated immune responses. Furthermore, the combination of different adjuvants like Alum and monophosphoryl lipid A (MPL) in the adjuvant system 04 (AS04) allowed for a reduction in antigen dose, faster production of antigen-specific antibodies, and longer duration of protective antibody titers compared to vaccines adjuvanted with aluminum salt alone (8).In the case of AS04, efficiently activated dendritic cells (DCs) stimulate polyfunctional antigen-specific CD4⁺ T cells. This effect was primarily dependent on MPL, while Alum was shown to prolong the MPL-induced cytokine response at the injection site, which is required for recruitment of antigen-presenting cells (9–11).Such designed adjuvant systems, containing different specific immune-modulating components, will be essential in current and future vaccine development. The risk for adverse effects, as observed upon Pandemrix vaccination, adjuvanted with an AS03 system (12, 13), needs to be as low as possible. In addition, adverse effects, which are potentially induced by adjuvants in healthy people, diminish the acceptance of vaccination within the population (14). Therefore, we need to increase our understanding of every distinct effect of single immunostimulatory components in adjuvants systems.Supported by the Ministry of Health, our research group at the Federal Institute for Vaccines and Biomedicines set up an in-depth immunological analysis of potential immune-modulating components. Herein, we performed a side-by-side comparison of various adjuvants using a human primary immune cell-based in vitro assay composed of monocyte-derived DCs (moDCs) and autologous peripheral blood lymphocytes (PBLs). DCs are the main target of adjuvants and react by secretion of proinflammatory cytokines, increased uptake, and MHC presentation of antigens as well as an enhanced expression of the costimulatory proteins.In our study, we aim to ascribe specific innate and adaptive immunogenic MoA to each adjuvant to facilitate the predictability of its potential to shape cellular immune responses and thus support the rational design of adjuvant-based vaccines. The adjuvant panel we investigated comprises second-generation and potential candidate adjuvants such as surface PRR ligands TDB (Mincle), Pam₃CSK₄ (Pam; TLR1/2), and MPL (TLR4); the endosomal PRR ligands Gardiquimod (GARD; TLR7), Imiquimod (IMQ; TLR7), and Resiquimod (R848; TLR7/8); and the saponin Quil A (Quil). In addition, first-generation adjuvants such as Al(OH)₃ and an oil-in-water emulsion AddaVax (ADX) are included. In light of the current success of mRNA-based vaccines against SARS-CoV-2, we hypothesize that their excellent antiviral immune protection does not only rely on the produced antigen but also on TLR7/8-dependent adjuvanticity, and we added lipid-complexed RNA to investigate this accordingly.In this study, several components were identified that induce full DC maturation, resulting in phenotypical and functional capacitation of DCs. In contrast to other single-component adjuvants, the TLR7 agonists GARD and IMQ only induced full DC maturation in the presence of PBLs. The tested adjuvants induced a highly specific response pattern, even when binding to similar or identical recognition receptors. It is the virtue of the approach taken in the current study to reveal the possibilities lying in the use of different agonists targeting the same receptor. Different components were additionally identified to have an antigen-dependent adjuvant effect on cells of the adaptive immune system. This effect was specific for the adjuvants Pam, MPL synthetic (MPL-s), and Al(OH)3, as they expanded FluM1-specific CD8⁺ T-cell populations in almost all of the tested donors. Using lipid-complexed single-stranded RNA as a surrogate for one of the recent mRNA vaccines, we could confirm that the cytokine profile induced by lipid-complexed RNA was almost identical to the pattern induced by synthetic TLR7/8 agonist R848. This supports the notion that the excellent antiviral immune protection that is observed with the innovative anti-SARS-CoV-2 RNA vaccines is in part due to the strong adjuvant activity of the single-stranded RNA component via TLR7/8.Thus, clearly showing that even similar adjuvants induce different immunogenic profiles, our data will prove to be important for vaccine developers to tailor and modulate the envisaged immune response as well as for drug regulators to support their understanding of adjuvanticity in the process of authorization of both safe and effective new vaccines.     

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.     

Toll样受体介导的天然免疫应答与常见病原体肺部感染之间的关系

Toll样受体家族是近年来发现的一类在宿主固有免疫及获得性免疫中起到关键作用的蛋白受体.越来越多的研究表明,Toll样受体与多种机体感染有关,包括肺部感染.本文就Toll样受体在肺部感染发病机制中的作用进行综述.     

Roles of liver innate immune cells in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease in the United States and other developed countries and is expected to increase in the next few years. Emerging data suggest that some patients with NAFLD may progress to nonalcoholic steatohepatitis (NASH), cirrhosis and even hepatocellular carcinoma. NAFLD can also promote the development and progression of disease in other organ systems, such as the cardiovascular and endocrine (i.e. diabetes) systems. Thus, understanding th...     

B and T lymphocyte attenuator inhibits LPS-induced endotoxic shock by suppressing Toll-like receptor 4 signaling in innate immune cells

Although innate immune responses are necessary for the initiation of acquired immune responses and the subsequent successful elimination of pathogens, excessive responses occasionally result in lethal endotoxic shock accompanied by a cytokine storm. B and T lymphocyte attenuator (BTLA), a coinhibitory receptor with similarities to cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed death (PD)-1, is expressed in not only B and T cells but also dendritic cells (DCs) and macrophages (Mϕs). Recently, several studies have reported that BTLA-deficient (BTLA^−/−) mice show enhanced pathogen clearance compared with WT mice in early phase of infections. However, the roles of BTLA expressed on innate cells in overwhelming and uncontrolled immune responses remain unclear. Here, we found that BTLA^−/− mice were highly susceptible to LPS-induced endotoxic shock. LPS-induced TNF-α and IL-12 production in DCs and Mϕs was significantly enhanced in BTLA^−/− mice. BTLA^−/− DCs also produced high levels of TNF-α on stimulation with Pam3CSK4 but not poly(I:C) or CpG, suggesting that BTLA functions as an inhibitory molecule on Toll-like receptor signaling at cell surface but not endosome. Moreover, BTLA^−/− DCs showed enhanced MyD88- and toll/IL-1R domain-containing adaptor inducing IFN (TRIF)-dependent signaling on LPS stimulation, which is associated with impaired accumulation of Src homology 2-containing protein tyrosine phosphatase in lipid rafts. Finally, we found that an agonistic anti-BTLA antibody rescued mice from LPS-induced endotoxic shock, even if the antibody was given to mice that had developed a sign of endotoxic shock. These results suggest that BTLA directly inhibits LPS responses in DCs and Mϕs and that agonistic agents for BTLA might have therapeutic potential for LPS-induced endotoxic shock.Septic shock is a life-threatening disease, which is caused by bacterial infection, especially with Gram-negative bacteria (1, 2). Toll-like receptor 4 (TLR4), one of representative pattern recognition receptors, recognizes LPS from Gram-negative bacteria and transduces signals in innate cells, such as macrophages (Mϕs) and dendritic cells (DCs), for the production of proinflammatory cytokines and chemokines (2–4). These innate responses are necessary for the initiation of acquired immune responses and subsequent successful elimination of bacteria. However, excessive innate immune responses occasionally result in a cytokine storm that is a potentially fatal immune reaction consisting of a positive feedback loop between highly elevated levels of various cytokines and immune cells, which leads to lethal endotoxic shock within a few days (1, 3, 5–8). However, lethal endotoxic shock is difficult to control by inhibitors for a particular cytokine (2, 7), and thus, novel therapeutic strategies for lethal endotoxic shock are desired.B and T lymphocyte attenuator (BTLA; CD272) is the third inhibitory coreceptor, which has been identified as an inhibitory coreceptor expressed on CD4⁺ T cells and B cells with similarities to CTLA-4 and PD-1 (9). Thereafter, accumulating evidence has revealed that BTLA is expressed on not only CD4⁺ T and B cells but also a wide range of hematopoietic cells, including CD8⁺ T cells, natural killer T cells, natural killer cells, Mϕs, and DCs at various levels (10). The ligand for BTLA is the TNF receptor family member Herpesvirus entry mediator (HVEM), which is broadly expressed on hematopoietic cells, including T cells, Mϕs, and DCs (10). Ligation of BTLA by HVEM induces the recruitment of SHP-1/SHP-2 and then attenuates cell activation (9–11). Analyses of BTLA-deficient (BTLA^−/−) mice have revealed that BTLA plays inhibitory roles in a variety of disease models, including experimental autoimmune encephalomyelitis (9), partially MHC-mismatched cardiac allograft (12), experimental colitis (13), and experimental hepatitis (14). We have also shown that the deficiency of BTLA spontaneously causes the breakdown of self-tolerance, resulting in the development of an autoimmune hepatitis-like disease and lymphocytic infiltration in multiple organs in aged mice (15). However, the administration of an agonistic anti-BLTA antibody has been shown to prevent graft-versus-host disease (16) and hapten-induced contact hypersensitivity (17). These results suggest that BTLA plays an important role in the homeostasis of acquired immune responses.In addition to the role of BTLA in acquired immune responses, recent studies have shown that BTLA also plays a role in immune responses against infectious pathogens. Sun et al. (18) have shown that BTLA^−/− mice exhibit significantly higher bacterial clearance compared with WT mice in the early phase of bacterial infection. Shubin et al. (19) have also shown that BTLA^−/− mice exhibited a higher rate of survival and protection from cecal ligation and puncture. Moreover, Adler et al. (20) have shown that BTLA^−/− mice exhibit strongly enhanced parasite clearance and that the increased clearance is seen before the onset of acquired immune responses. These findings suggest that BTLA is involved in the clearance of pathogens in the early phase of immune responses and that BTLA expressed on innate cells might be involved in the process. However, the role of BTLA in overwhelming and uncontrolled immune responses that lead to endotoixic shock remains unclear.In this study, we examined the role of BTLA in innate immune responses and found that BTLA signaling inhibited LPS-induced endotoxic shock and proinflammatory cytokine production from innate cells. We also found that BTLA signaling inhibited both LPS-induced MyD88- and TRIF-dependent pathways in DCs, possibly by inducing the recruitment of SHP-2 into lipid rafts. We also showed that an agonistic anti-BTLA antibody had therapeutic potential for endotoxic shock. Our results highlight the importance of BTLA in innate immune responses.     

微载体培养人肝细胞及肝非实质细胞

目的研究用微载体培养人肝细胞及肝非实质细胞的方法．方法采用体外简易两步灌流和差速离心法获取胎肝细胞和肝非实质细胞，在综合限定条件下进行微载体ｃｙｔｏｄｅｘ３粘附培养，并对培养肝细胞的形态及合成葡萄糖和白蛋白的功能进行动态测定．结果分离肝细胞及肝非实质细胞在接种时即呈明显的聚集倾向，将其与微载体混合振荡孵育２０ｍｉｎ后，二者极易相粘附．在被覆聚羟乙基异丁烯酸的培养瓶内，使用激素限定条件培养液培养约４８ｈ，典型的多细胞聚集球形体得以形成．该形态特征以及白蛋白、葡萄糖合成能力可保持１月结论使用微载体培养人肝细胞和肝非实质细胞具有多种优点，有广泛的应用价值．     

Pattern recognition receptors and the innate immune response to viral infection

The innate immune response to viral pathogens is critical in order to mobilize protective immunity. Cells of the innate immune system detect viral infection largely through germline-encoded pattern recognition receptors (PRRs) present either on the cell surface or within distinct intracellular compartments. These include the Toll-like receptors (TLRs), the retinoic acid-inducble gene I-like receptors (RLRs), the nucleotide oligomerization domain-like receptors (NLRs, also called NACHT, LRR and PYD domain proteins) and cytosolic DNA sensors. While in certain cases viral proteins are the trigger of these receptors, the predominant viral activators are nucleic acids. The presence of viral sensing PRRs in multiple cellular compartments allows innate cells to recognize and quickly respond to a broad range of viruses, which replicate in different cellular compartments. Here, we review the role of PRRs and associated signaling pathways in detecting viral pathogens in order to evoke production of interferons and cytokines. By highlighting recent progress in these areas, we hope to convey a greater understanding of how viruses activate PRR signaling and how this interaction shapes the anti-viral immune response.     

Use of recombinant adeno-associated viral vectors as a tool for labeling bone marrow cells

We have tested the feasibility of using recombinant adeno-associated virus (rAAV) vectors as a tool for labeling bone marrow (BM) cells in vivo. We infected BM cells of donor FVB mice with rAAV vectors containing the lacZ gene for 2 h. We then injected the rAAV-infected cells to lethally irradiated-recipient FVB mice. Peripheral blood (PB), BM and spleen harvested at 4 weeks after BM transplant (BMT) demonstrated stable engraftment in beta-galactosidase (beta-gal) expression. In contrast, Dil-labeling displayed only a faint signal 4 weeks after BMT. To analyze the kinetics of BM cells, we injected vascular endothelial growth factor (VEGF), which promotes mobilization of BM cells. Administration of VEGF protein significantly increased the rAAV-mediated beta-gal expression in PB and BM of recipient mice. Moreover, when myocardial infarction was induced in BMT mice, the ischemic area exhibited significant beta-gal staining in rAAV-labeled BMT group. rAAV vectors programmed stable transduction in BM cells in vivo through rapid infection. rAAV appears to represent a useful vector for labeling BM cells ex vivo prior to BMT for analysis of cardiovascular therapeutic purposes.     

Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA

Long-term expression of coagulation factor IX (FIX) has been observed in murine and canine models following administration of recombinant adeno-associated viral (rAAV) vectors into either the portal vein or muscle. These studies were designed to evaluate factors that influence rAAV-mediated FIX expression. Stable and persistent human FIX (hFIX) expression (> 22 weeks) was observed from 4 vectors after injection into the portal circulation of immunodeficient mice. The level of expression was dependent on promoter with the highest expression, 10% of physiologic levels, observed with a vector containing the cytomegalovirus (CMV) enhancer/beta-actin promoter complex (CAGG). The kinetics of expression after injection of vector particles into muscle, tail vein, or portal vein were similar with hFIX detectable at 2 weeks and reaching a plateau by 8 weeks. For a given dose, intraportal administration of rAAV CAGG-FIX resulted in a 1.5-fold or 4-fold higher level of hFIX compared to tail vein or intramuscular injections, respectively. Polymerase chain reaction analysis demonstrated predominant localization of the rAAV FIX genome in liver and spleen after tail vein injection with a higher proportion in liver after portal vein injection. Therapeutic levels of hFIX were detected in the majority of immunocompetent mice (21 of 22) following intravenous administration of rAAV vector without the development of anti-hFIX antibodies, but hFIX was not detected in 14 immunocompetent mice following intramuscular administration, irrespective of strain. Instead, neutralizing anti-hFIX antibodies were detected in all the mice. These observations may have important implications for hemophilia B gene therapy with rAAV vectors.