Ling-Jun Ho

Dengue Virus Type 2 Antagonizes IFN-α but Not IFN-γ Antiviral Effect via Down-Regulating Tyk2-STAT Signaling in the Human Dendritic Cell

The immunopathogenesis mechanism of dengue virus (DV) infection remains elusive. We previously showed that the target of DV in humans is dendritic cells (DCs), the primary sentinels of immune system. We also observed that despite the significant amount of IFN-α induced; DV particles remain massively produced from infected DCs. It suggests that DV may antagonize the antiviral effect of IFN-α. Recent work in animal studies demonstrated the differential critical roles of antiviral cytokines, namely IFN-α/IFN-β and IFN-γ, in blocking early viral production and in preventing viral-mediated disease, respectively. In this study, we examined the effects of IFN-α and IFN-γ in DV infection of monocyte-derived DCs. We showed that the preinfection treatment with either IFN-α or IFN-γ effectively armed DCs and limited viral production in infected cells. However, after infection, DV developed mechanisms to counteract the protection from lately added IFN-α, but not IFN-γ. Such a selective antagonism on antiviral effect of IFN-α, but not IFN-γ, correlated with down-regulated tyrosine-phosphorylation and DNA-binding activities of STAT1 and STAT3 transcription factors by DV. Furthermore, subsequent studies into the underlying mechanisms revealed that DV attenuated IFN-α-induced tyrosine-phosphorylation of Tyk2, an upstream molecule of STAT activation, but had no effect on expression of both IFN-α receptor 1 and IFN-α receptor 2. Moreover, DV infection by itself could activate STAT1 and STAT3 through IFN-α-dependent and both IFN-α-dependent and IFN-α-independent mechanisms, respectively. These observations provide very useful messages with physiological significance in investigation of the pathogenesis, the defense mechanisms of human hosts and the therapeutic considerations in DV infection.

Chinese herbs as immunomodulators and potential disease-modifying antirheumatic drugs in autoimmune disorders.

Autoimmune diseases are a group of illnesses with multiple organ involvement. The prototype of this group of disorders is rheumatoid arthritis (RA) that aside from systemic organ involvement mainly presents with progressive destruction of many joints. Both activation and defective apoptosis of immune effector cells like T and B lymphocytes and macrophages play critical roles in the pathogenesis of autoimmune disorders. Current therapy for autoimmune diseases recommends a combination of several disease-modifying antirheumatic drugs (DMARDs) that preserve different immunomodulatory mechanisms. Because of limited success in prevention of RA joint destruction for currently available DMARDs, the development of more effective and less toxic DMARDs has been one of the major goals for pharmaceutical companies. The introduction of leflunomide and anti-tumor necrosis factor alpha therapies to the market recently serves as examples. In this context, the experience from ancient Chinese medicine gives an alternative consideration looking for potential DMARDs. Two commonly prescribed Chinese antirheumatic herbs are Tripterygium wilfordii hook f (TWHf) and tetrandrine (Tet) that preserve both anti-inflammatory and immunosuppressive effects. Importantly, the TWHf- or Tet-mediated immunomodulatory mechanisms are evidently different from the known DMARDs. The synergistic effects have also been demonstrated between these two Chinese antirheumatic herbs and DMARDs like FK506, cyclosporin and possibly chloroquine. Another potential Chinese herb for this consideration is Ginkgo biloba. This review summarizes evidence-based in vivo and in vitro studies on Chinese herbs as immunomodulators and potential DMARDs.

Plant alkaloid tetrandrine downregulates IκBα kinases-IκBα-NF-κB signaling pathway in human peripheral blood T cell

Plant alkaloid tetrandrine (Tet), purified from Chinese herb Han‐Fang Chi, is a potent immunomodulator used to treat rheumatic disorders, silicosis and hypertension in mainland China. We previously demonstrated that Tet effectively suppresses cytokine production and proliferation of CD28‐costimulated T cells. In the present study, we investigated the possible involvement of nuclear factor kappa B (NF‐κB) transcription factors, critical in CD28 costimulation, in Tet‐mediated immunosuppression in human peripheral blood T cells. We showed that Tet inhibited NF‐κB DNA‐binding activities induced by various stimuli, including CD28 costimulation. At equal molar concentrations, Tet was as strong as methotrexate in suppressing CD28‐costimulated NF‐κB activities. Since Tet itself did not affect NF‐κB binding to its corresponding DNA sequence, the results suggested that Tet might regulate NF‐κB upstream signaling molecules. Further studies demonstrated that Tet could prevent the degradation of IκBα and inhibit nuclear translocation of p65 by blocking IκBα kinases α and β activities. In addition, the activation of mitogen‐activated protein kinases such as c‐jun N‐terminal kinase, p38 and extracellular signal‐regulated kinase and activator protein‐1 DNA‐binding activity were all downregulated by Tet. Transfection assays performed in purified human peripheral blood T cells also confirmed the inhibition of NF‐κB transcriptional activity by Tet. When four Tet analogues were readily compared, dauricine appeared to preserve the most potent inhibition on CD28‐costimulated but not on H2O2‐induced NF‐κB DNA‐binding activities. Our results provide the molecular basis of immunomodulation of Tet for being a potential disease‐modifying antirheumatic drug in the therapy of autoimmune disorders like rheumatoid arthritis.

Western and Chinese Antirheumatic Drug-Induced T Cell Apoptotic DNA Damage Uses Different Caspase Cascades and Is Independent of Fas/Fas Ligand Interaction

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.

Plant alkaloid tetrandrine and its analog block CD28-costimulated activities of human peripheral blood T cells: potential immunosuppressants in transplantation immunology.

BACKGROUND T lymphocyte activation mediated by CD28 costimulation plays a critical role in graft rejection. Plant alkaloid tetrandrine, purified from a Chinese antirheumatic herb, is a potent immunosuppressant. Here, we examined its effects on several CD28-costimulated T-cell activities. In addition, such effects were readily compared with the effects of three tetrandrine analogs. METHODS T lymphocytes were purified from whole blood by negative selection. The stimuli that mimic CD28 costimulation included both anti-CD3 + anti-CD28 monoclonal antibody and PMA+anti-CD28 monoclonal antibody. The determination of CD28-costimulated cell proliferation was performed by tritium uptake, cytokine production by ELISA, cell surface interleukin 2Ra and CD69 expression by flow cytometry, and mixed leukocyte reaction by tritium uptake. Drug cytotoxicity was determined by trypan blue exclusion, propidium iodide staining, and MTT colorimetric assays. RESULTS Tetrandrine inhibited CD28-costimulated T-cell proliferation and cytokine production through a mechanism different from that of cyclosporine. In addition, tetrandrine down-regulated both T helper 1 and T helper 2 cytokine production in CD4+ and CD8+ T-cell subpopulations. By examining cytokine production and T-cell activation marker expression, we further demonstrated that, among tetrandrine and its analogs tested, dauricine was the most potent suppressor of CD28-costimulated T-cell activities. Furthermore, the different immunosuppressive activities of these compounds were not associated with their cytotoxic capacities. Finally, the unparalleled inhibitory potency of dauricine on both mixed leukocyte reaction and CD28-costimulated T-cell proliferation suggests that dauricine preferentially targeted CD28-costimulated T-cell activities. CONCLUSIONS This is the first report to show that tetrandrine and its analogs potently inhibited both PMA+CD28-costimulated and CD3 + CD28-costimulated activation of human peripheral blood T cells. Based upon their structural similarity and different immunosuppressive potency, these in vitro data also provide very useful information for further identification and development of more potent and less toxic immunosuppressants to achieve transplantation success.

Aspirin differentially regulates endotoxin-induced IL-12 and TNF-α production in human dendritic cells

OBJECTIVE In the development of autoimmune diseases, dendritic cells (DC) play critical roles. Here, we examined the effect of aspirin on lipopolysaccharide (LPS)-induced DC activation. METHODS The monocyte-derived DC were established. The cytokine production was measured by ELISA, reverse transcriptase/polymerase chain reaction, or intracellular staining analyzed by flow cytometry. The expression of cell surface molecules was determined by flow cytometry. RESULTS Aspirin inhibited LPS-induced DC maturation and costimulatory molecules expression. Aspirin, at therapeutic concentrations, also decreased LPS-induced IL-12 and IL-10 production. In contrast, the LPS-induced TNF-alpha production was enhanced by aspirin. The differential effects of aspirin on IL-12 and TNF-alpha production may not be due to down-regulation of cyclooxygenase activities. CONCLUSION The various effects of aspirin on LPS-stimulated DC may influence the understanding of the diverse immunomodulatory mechanisms of this anti-inflammatory drug.Objective : In the development of autoimmune diseases, dendritic cells (DC) play critical roles. Here, we examined the effect of aspirin on lipopolysaccharide (LPS)-induced DC activation. Methods : The monocyte-derived DC were established. The cytokine production was measured by ELISA, reverse transcriptase/ polymerase chain reaction, or intracellular staining analyzed by flow cytometry. The expression of cell surface molecules was determined by flow cytometry. Results : Aspirin inhibited LPS-induced DC maturation and costimulatory molecules expression. Aspirin, at therapeutic concentrations, also decreased LPS-induced IL-12 and IL-10 production. In contrast, the LPS-induced TNF- f production was enhanced by aspirin. The differential effects of aspirin on IL-12 and TNF- f production may not be due to down-regulation of cyclooxygenase activities. Conclusion : The various effects of aspirin on LPS-stimulated DC may influence the understanding of the diverse immunomodulatory mechanisms of this anti-inflammatory drug.

Plant alkaloid tetrandrine downregulates protein kinase C-dependent signaling pathway in T cells

Tetrandrine, a purified traditional Chinese medicinal herb that acts as an immunosuppressant and a Ca2+ channel blocker, has been clinically used to treat patients with arthritis, silicosis and hypertension. Since T cells play a critical role as autoreactive and pathogenic population in autoimmune diseases, in this study, we examined the immunosuppressive effect of tetrandrine on human peripheral blood T cells. We showed that tetrandrine inhibited phorbol 12-myristate 13-acetate (PMA) + ionomycin-induced T cell proliferation, interleukin-2 secretion and the expression of the T cell activation antigen, CD71. Further investigation of the molecular mechanism demonstrated that tetrandrine inhibited the expression of the protein kinase C-dependent interleukin-2 receptor alpha chain and CD69 but not the expression of the Ca2+-dependent CD40 ligand and CD69. Interestingly, when tetrandrine and cyclosporin A were added together, significant synergism in the suppression of T cell activation was observed. Moreover, of the several tetrandrine analogues studied, hernandezine was the most potent inhibitor of protein kinase C signaling events. These results also suggest that the protein kinase C-inhibitory capacity of tetrandrine and its analogues may not be associated with their function as Ca2+ channel blockers. Lastly, we showed that, within therapeutic concentrations, tetrandrine and its analogues could induce cellular apoptosis, which is defective in autoimmune diseases. In conclusion, our findings provide novel information about the molecular mechanism of the immunosuppressive effect of tetrandrine and its analogues in human peripheral blood T cells.

Biological effects of interleukin-6: Clinical applications in autoimmune diseases and cancers

Interleukin-6 (IL-6) is a pro-inflammatory cytokine involved in the pathogenesis of various autoimmune and chronic inflammatory diseases. Binding of IL-6 to its receptor (IL-6R) initiates both classical- and trans-signaling pathways. A number of autoimmune diseases are characterized by overproduction of IL-6. Tocilizumab, a humanized monoclonal antibody against IL-6R, blocks IL-6-mediated signaling and has been approved for the treatment of rheumatoid arthritis and Castlemans disease. IL-6 levels are also upregulated in various tumors, and the levels of circulating IL-6 are associated with prognosis in cancer patients. The major issues covered in this commentary include (1) how IL-6-mediated biological effects may lead to the pathogenesis of autoimmune diseases and cancers, (2) the rationale of developing anti-IL-6 strategies for therapeutic purposes, (3) recent advances in anti-IL-6 therapeutics (clinical benefits and adverse events), (4) current knowledge about clinical trials evaluating newly emerging anti-IL-6 treatments, (5) strategies to improve anti-IL-6 therapeutics from both basic and clinical aspects. This commentary provides a useful overview of the role of IL-6 in both autoimmune diseases and cancers from the laboratory as well as clinical perspectives.

Infection of Human Dendritic Cells by Dengue Virus Activates and Primes T Cells Towards Th0‐Like Phenotype Producing Both Th1 and Th2 Cytokines

Dengue viruses (DV) infection is an important public health issue all over the world. Although the pathogenesis remains unclear, the overwhelmingly triggered immune responses have been consistently observed. Recently, we and other researchers demonstrated that the natural hosts for DV are dendritic cells (DC), the primary sentinels of immune system. In light of the significance of T cells in dengue virus pathogenesis, here, we examine the possible consequences of DC‐T cell interaction that is supposed to be happening in lymphoid tissues after infection. We showed that DV‐infected DC induced the interacting T cells to proliferate, to produce interleukin‐2 as well as to express activation markers on cell surface. Compared to mock‐infected DC, the infection of DC by DV also induced T cells to produce interleukin‐4, interleukin‐10 and interferon‐gamma, a cytokine pattern suggesting Th0 phenotype. Such an effect was either totally abolished or greatly reduced when DV were pre‐inactivated with heat or ultraviolet before infection. In addition, we demonstrated that such a Th0 phenotype shift of T cells was affected neither by different dosages of viruses that infected DC nor by different durations of DC‐T cell interaction. Our results provide a basic support for clinical observations and may be of help in understanding the pathogenesis of DV infection.

Triggering of DC migration by dengue virus stimulation of COX-2-dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation.

A term “bone‐breaking fever” is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX‐prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX‐2 and the production of prostaglandin E2 (PGE2) in DC, and stimulated the DNA binding of NF‐κB and the kinase activity of both IκBα kinase (IKK) α and β. DV infection also activated MAPK and AP‐1 signaling. Both IκBα kinase‐NF‐κB and MAPK‐AP‐1 were upstream of COX‐2 activation. Our investigation into the significance of COX‐2‐PGE2 pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX‐2 or MAPK activity suppresses DV‐induced DC migration. Our data also suggest that PGE2 can induce CCR7 expression on DC and that antagonists of the PGE2 receptors EP2 and EP4 suppress DV‐induced DC migration. We further show that the increased CCR7 expression was observed in both DV‐infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX‐2 synthesis in DV‐infected DC but also the autocrine action of PGE2 on the migration of DV‐infected DC.