ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses.

Zinc is a trace element that is essential for immune responses. Therefore, changes in cellular zinc levels in specific immune cells may influence inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). However, the regulation of zinc mobilization in immune cells and its role in the pathogenesis of RA are not fully understood. Thus, we investigated the roles of zinc transporters in RA pathogenesis. We demonstrated that ZIP8 was specifically upregulated in CD4+ T cells that infiltrated the inflamed joint and that ZIP8 deficiency in CD4+ T cells abrogated collagen-induced arthritis. ZIP8 deficiency dramatically affected zinc influx in effector T cells and profoundly reduced T cell receptor (TCR)-mediated signaling, including NF-κB and MAPK signaling, which are pathways that are involved in T helper (Th) 17 cell differentiation. Taken together, our findings suggest that ZIP8 depletion in CD4+ T cells attenuates TCR signaling due to insufficient cellular zinc, thereby reducing the function of effector CD4+ T cells, including Th17 cells. Our results also suggest that targeting ZIP8 may be a useful strategy to inhibit RA development and pathogenesis.

Discovery of Novel Pyrimidine-Based Capsid Assembly Modulators as Potent Anti-HBV Agents.

Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.

Development of a mass spectrometric screening assay for hepatitis B virus entry inhibitors.

Sodium taurocholate cotransporting polypeptide (NTCP) involved in bile acid transport in the liver is an entry receptor of hepatitis B virus (HBV). In the present study, we introduce a mass spectrometric screening assay for targeting HBV entry inhibitors that can reduce NTCP transporter activity by employing taurocholic acid (TCA) labeled with stable isotope (2,2,4,4-d4-TCA, d4-TCA) and NTCP-overexpressing human liver cancer cell lines such as HepG2 and Huh-7. The accuracy and reliability of the proposed mass spectrometric NTCP activity assay have been validated with known HBV inhibitors including cyclosporine A (CsA) and pre-S1 peptide (PreS/2-48Myr or myrcludex B analog) that suppress the entry of HBV into hepatocytes by targeting NTCP. For the inhibitor screening assay, NTCP-overexpressing HepG2 or Huh-7 cells are treated with either a combination of TCA and an inhibitor (CsA or PreS/2-48Myr) or d4-TCA alone to serve as a reference. The activity of an HBV inhibitor is determined by relative quantification between TCA and d4-TCA in a 1:1 mixture of inhibitor-treated cells and untreated control cells using liquid chromatography-mass spectrometry. With our new approach, the half maximal inhibitory concentration (IC50) values for CsA and PreS/2-48Myr have been determined at micromolar and nanomolar concentrations, respectively, which is consistent with the previous results obtained with other conventional HBV entry inhibitor assay methods. Our assay method does not require HBV infection or radioactive 3H-TCA and provides a facile way to identify viral entry inhibitors via measuring bile acid transport activity of NTCP.

Ciclopirox inhibits Hepatitis B Virus secretion by blocking capsid assembly.

Chronic hepatitis B virus (HBV) infection can cause cirrhosis and hepatocellular carcinoma and is therefore a serious public health problem. Infected patients are currently treated with nucleoside/nucleotide analogs and interferon α, but this approach is not curative. Here, we screen 978 FDA-approved compounds for their ability to inhibit HBV replication in HBV-expressing HepG2.2.15 cells. We find that ciclopirox, a synthetic antifungal agent, strongly inhibits HBV replication in cells and in mice by blocking HBV capsid assembly. The crystal structure of the HBV core protein and ciclopirox complex reveals a unique binding mode at dimer-dimer interfaces. Ciclopirox synergizes with nucleoside/nucleotide analogs to prevent HBV replication in cells and in a humanized liver mouse model. Therefore, orally-administered ciclopirox may provide a novel opportunity to combat chronic HBV infection by blocking HBV capsid assembly.

Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1ß.

Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell populations within tumor tissues colonized by E. coli or attenuated Salmonellae. CT26 tumor-bearing mice were treated with two different bacterial strains: S. typhimurium defective in ppGpp synthesis (ΔppGpp Salmonellae) or wild-type E. coli MG1655. Cytokine profiles and immune cell populations in tumor tissue colonized by these two bacterial strains were examined at two time points based on the pattern of tumor growth after ΔppGpp Salmonellae treatment: 1) when tumor growth was suppressed ('suppression stage') and 2) when they began to re-grow ('re-growing stage'). The levels of IL-1ß and TNF-α were markedly increased in tumors colonized by ΔppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1ß and TNF-α returned to normal level when the tumors started to re-grow. To identify the immune cells primarily responsible for Salmonellae-mediated tumor suppression, we examined the major cell types that produce IL-1ß and TNF-α. We found that macrophages and dendritic cells were the main producers of TNF-α and IL-1ß. Inhibiting IL-1ß production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1ß or TNF-α in conjunction with Salmonella therapy. These findings suggested that IL-1ß and TNF-α play important roles in Salmonella-mediated cancer therapy. A better understanding of host immune responses in Salmonella therapy may increase the success of a given drug, particularly when various strategies are combined with bacteriotherapy.