Apoptosis and proinflammatory cytokine responses of primary mouse microglia and astrocytes induced by human H1N1 and avian H5N1 influenza viruses

Patients with an influenza virus infection can be complicated by acute encephalopathy and encephalitis. To investigate the immune reactions involved in the neurocomplication, mouse microglia and astrocytes were isolated, infected with human H1N1 and avian H5N1 influenza viruses, and examined for their immune responses. We observed homogeneously distributed viral receptors, sialic acid （SA）-a2,3-Galactose （Gal） and SA-a2,6-Gal, on microglia and astrocytes. Both viruses were replicative and productive in microglia and astrocytes. Virus-induced apoptosis and cytopathy in infected cells were observed at 24 h post-infection （p.i.）. Expression of IL-1β, IL-6 and TNF-a mRNA examined at 6 h and 24 h p.i. was up-regulated, and their expression levels were considerably higher in H5N1 infection. The amounts of secreted proinflammatory IL-1β, IL-6 and TNF-a at 6 h and 24 h p.i. were also induced, with greater induction by H5N1 infection. This study is the first demonstration that both human H1N1 and avian H5N1 influenza viruses can infect mouse microglia and astrocytes and induce apoptosis, cytopathy, and proinflammatory cytokine production in them in vitro. Our results suggest that the direct cellular damage and the consequences of immunopathological injury in the CNS contribute to the influenza viral pathogenesis. Cellular ＆ Molecular Immunology.     

Interleukin 17-Producing γδ T Cells Increased in Patients with Active Pulmonary Tuberculosis

Although it has been known that γδ T cells may play an important role in the immune response to infection of Mycobacterium tuberculosis （M. tb）, the mechanisms by which the γδ T cells participate in the innate and/or acquired immunity to tuberculosis （TB） have not been full elucidated. In the present study, 27 patients with active pulmonary TB and 16 healthy donors （HD） were performed. We found that proportion of IL-17-producing cells among lymphocyte was similar between TB patients and HD, whereas the proportions of γδ T cells in IL-17-producing cells （59.2%） and IL-17-producing cells in γδ T cells （19.4%） in peripheral blood were markedly increased in TB patients when compared to those in HD （43.9% and 7.7%, respectively）. In addition, the proportions of IFN-T-producing γδ T cells in TB patients were obviously lower than that in HD. Upon re-stimulated with M. tb heat-treated antigen （M. tb-HAg） in vitro, fewer IL-17-producing γδ T cells were generated from HD and TB patients, whereas IFN-T-producing γδ T cells were increased in TB patients compared to that in HD. Our findings in TB patients and healthy human were consistent with other murine investigation that the IL-17- producing γδ T cells were main source of IL-17 in mouse model of BCG infection, suggesting that γδ T cells might be involved in the formation of tubercular granuloma in pulmonary TB patients, but need further identification.     

Selective Function of PKC-θ in T cells

T cell activation is a critical process in initiating adaptive immune response since only through this process the naive antigen specific T cells differentiate into armed effector T cells that mediate the actual immune response. During T cell activation, naive T cells undergo clonal expansion and acquire the capability to kill target cells infected with pathogens or produce cytokines essential for regulating immune response. Inappropriate activation or inactivation of T cells leads to autoimmunity or severe immunodeficiencies. PKC-θ is selectively expressed in T cells and required for mediating T cell activation process. Mice deficient in PKC-θ exhibit defects in T cell activation, survival and activation-induced cell death. PKC-θ selectively translocates to immunological synapse and mediates the signals required for activation of NF-κB, AP1 and NFAT that are essential for T cell activation. Furthermore, PKC-θ^-│- mice displayed multiple defects in the development of T cell-mediated immune responses in vivo. PKC-θ is thus a critical molecule that regulates T cell function at multiple stages in T cell-mediated immune responses in vivo.     

Interleukin 17-Producing γδT Cells Increased in Patients with Active Pulmonary Tuberculosis

Although it has been known that y8 T cells may play an important role in the immune response to infection of Mycobacterium tuberculosis (M. tb), the mechanisms by which the T8 T cells participate in the innate and/or acquired immunity to tuberculosis (TB) have not been full elucidated. In the present study, 27 patients with active pulmonary TB and 16 healthy donors (HD) were performed. We found that proportion of IL-17-producing cells among lymphocyte was similar between TB patients and HD, whereas the proportions of γδ T cells in IL-17- producing cells (59.2%) and IL-17-producing cells in γδ T cells (19.4%) in peripheral blood were markedly increased in TB patients when compared to those in HD (43.9% and 7.7%, respectively). In addition, the proportions of IFN-γ-producing γδ T cells in TB patients were obviously lower than that in HD. Upon re-stimulated with M. tb heat-treated antigen (M. tb-HAg) in vitro, fewer IL-17-producing γδ T cells were generated from HD and TB patients, whereas IFN-γ-producing γδ T cells were increased in TB patients compared to that in HD. Our findings in TB patients and healthy human were consistent with other murine investigation that the IL-17- producing γδ T cells were main source of IL-17 in mouse model of BCG infection, suggesting that γδ T cells might be involved in the formation of tubercular granuloma in pulmonary TB patients, but need further identification. Cellular & Molecular Immunology. 2008;5(3):203-208.     

The Plasticity of γδ T Cells： Innate Immunity,Antigen Presentation and New Immunotherapy

Several signals influence dendritic cell （DC） functions and consequent the immune responses to infectious pathogens. Our recent findings provide a new model of intervention on DCs implicating human γδ T cell stimuli. Vγ/9Vδ2 T cells represent the major subset of circulating human γδ T cells and can be activated by non-peptidic molecules derived from different microorganisms or abnormal metabolic routes. With activated-Vγ/9Vδ2 T cell co-culture, immature DCs acquire features of mature DCs, such as increasing the migratory activity, up-regulating the chemokine receptors, and triggering the Thl immune response. Similar to the NK-derived signals, DC activation is mediated by soluble factors as well as cell-to-cell contact. Many non-peptidic molecules including nitrogen- containing bisphosphonates and pyrophosphomonoester drugs, can stimulate the activity of Vγ/9Vδ2 T cells in vitro and in vivo. The relatively low in vivo toxicity of many of these drugs makes possible novel vaccine and immune-based strategies against infectious diseases.     

The role of chemokines in acute and chronic hepatitis C infection

Hepatitis C imposes a significant burden on global healthcare. Chronic infection is associated with progressive inflammation of the liver which typically manifests in cirrhosis, organ failure and cancer. By virtue of elaborate evasion strategies, hepatitis C virus （HCV） succeeds as a persistent human virus. It has an extraordinary capacity to subvert the immune response enabling it to establish chronic infections and associated liver disease. Chemokines are low molecular weight chemotactic peptides that mediate the recruitment of inflammatory cells into tissues and back into the lymphatics and peripheral blood. Thus, they are central to the temporal and spatial distribution of effector and regulatory immune cells. The interactions between chemokines and their cognate receptors help shape the immune response and therefore, have a major influence on the outcome of infection. However, chemokines represent a target for modulation by viruses including the HCV. HCV is known to modulate chemokine expression in vitro and may therefore enable its survival by subverting the immune response in vivo through altered leukocyte chemotaxis resulting in impaired viral clearance and the establishment of chronic low-grade inflammation. In this review, the roles of chemokines in acute and chronic HCV infection are described with a particular emphasis placed on chemokine modulation as a means of immune subversion. We provide an in depth discussion of the part played by chemokines in mediating hepatic fibrosis while addressing the potential applications for these chemoattractants in prognostic medicine.     

TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells

The combination of immunotherapy and chemotherapy is regarded as a promising approach for the treatment of certain types of cancer. However, the underlying mechanisms need to be fully investigated to guide the design of more efficient protocols for cancer chemoimmunotherapy. It is well known that danger-associated molecular patterns （DAMPs） can activate immune cells, including dendritic cells （DCs）, via Toll-like receptors （TLRs）; however, the role of DAMPs released from chemical drug-treated tumor cells in the activation of the immune response needs to be further elucidated. Here, we found that colorectal cancer （CRC） cells treated with oxaliplatin （OXA） and/or 5-fluorouracil （5-Fu） released high levels of high-mobility group box 1 （HMGB1） and heat shock protein 70 （HSP70）. After OXA/5-Fu therapy, the sera of CRC patients also exhibited increased levels of HMGB1 and HSP70, both of which are well-known DAMPs. The supernatants of dying CRC cells treated with OXA/5-Fu promoted mouse and human DC maturation, with upregulation of HLA-DR, CD80 and CD86 expression and enhancement of IL-lp, TNF-a, MIP-la, MIP-lp, RANTES and IP-IO production. Vaccines composed of DCs pulsed with the supernatants of chemically stressed CRC cells induced a more significant IFN-y-producing Thl response both in vitroand in vivo. However, the supernatants of chemically stressed CRC cells failed to induce phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating an essential role of TLR4 in DAMP-induced DC maturation and activation. Furthermore, pulsing with the supernatants of chemically stressed CRC cells did not efficiently induce an IFN-y-producing Thl response in TLR4-deficient DCs. Collectively, these results demonstrate that DAMPs released from chemically stressed cancer cells can activate DCs viaTLR4 and enhance the induction of an anti-tumor T-cell immune response, delineating a clinically relevant immuno-adjuvant pathway triggered by DAMPs.     

A Functional Type I Interferon Pathway Drives Resistance to Cornea Herpes Simplex Virus Type 1 Infection by Recruitment of Leukocytes

Type I interferons are critical antiviral cytokines produced following herpes simplex virus type-1 (HSV-1) infection that act to inhibit viral spread.In the present study,we identify HSV-infected and adjacent uninfected corneal epithelial cells as the source of interferon-α.We also report mice deficient in the A1 chain of the type I IFN receptor (CD118-/) are extremely sensitive to ocular infection with low doses (100 PFU) of HSV-1 as seen by significantly elevated viral titers in the cornea compared to wild type (WT) controls.The enhanced susceptibility correlated with a loss of CD4 + and CD8 + T cell recruitment and aberrant chemokine production in the cornea despite mounting an adaptive immune response in the draining mandibular lymph node of CD118-/mice.Taken together,these results highlight the importance of IFN production in both the innate immune response as well as eliciting chemokine production required to facilitate adaptive immune cell trafficking.     

The Plasticity of γδT Cells: Innate Immunity, Antigen Presentation and New Immunotherapy

Several signals influence dendritic cell (DC) functions and consequent the immune responses to infectious pathogens. Our recent findings provide a new model of intervention on DCs implicating human γδ T cell stimuli. Vγ9Vδ2 T cells represent the major subset of circulating human γδ T cells and can be activated by non-peptidic molecules derived from different microorganisms or abnormal metabolic routes. With activated-Vγ9Vδ2 T cell co-culture, immature DCs acquire features of mature DCs, such as increasing the migratory activity, up-regulating the chemokine receptors, and triggering the Thl immune response. Similar to the NK-derived signals, DC activation is mediated by soluble factors as well as cell-to-cell contact. Many non-peptidic molecules including nitrogen- containing bisphosphonates and pyrophosphomonoester drugs, can stimulate the activity of Vγ9Vδ2 T cells in vitro and in vivo. The relatively low in vivo toxicity of many of these drugs makes possible novel vaccine and immune-based strategies against infectious diseases. Cellular & Molecular Immunology. 2008;5(3):161-170.     

Death-associated protein kinase 1 is an IRF3/7-interacting protein that is involved in the cellular antiviral immune response

Interferon regulatory factor （IRF） 7 has been demonstrated to be a master regulator of virus-induced type I interferon production （IFN）, and it plays a central role in the innate immune response against viruses. Here, we identified death-associated protein kinase 1 （DAPK1） as an IRF7-interacting protein by tandem affinity purification （TAP）. Viral infection induced DAPKI-IRF7 and DAPKI-IRF3 interactions and overexpression of DAPK1 enhanced virus-induced activation of the interferon-stimulated response element （ISRE） and IFN-p promoters and the expression of the IFNB1 gene. Knockdown of DAPK1 attenuated the induction of IFNB1 and RIG.lexpression triggered by viral infection or I FN-p, and they were enhanced by viral replication. In addition, viral infection or IFN-p treatment induced the expression of DAPK1. IFN-p treatment also activated DAPK1 by decreasing its phosphorylation level at serine 308. Interestingly, the involvement of DAPK1 in virus-induced signaling was independent of its kinase activity. Therefore, our study identified DAPK1 as an important regulator of the cellular antiviral response.     

T cell vaccination as an immunotherapy for autoimmune diseases

Immunization with inactivated autoreactive T cells(T cell vaccination) selected from individual's own T cellrepertoire provides a unique in vivo setting for testing immune regulation that is known to involve interactionsof a variety of related surface molecules(1).It induces regulatory immune responses that closely resemble thein vivo situation where the immune system is challenged by clonal activation and expansion of given T cellpopulations in various autoimmune diseases.T cell vaccination provides a powerful means of eliciting naturalreactions of the immune system in response to clonal expansion of T cells,which can be used as a therapeuticapproach to suppress or eliminate specific pathogenic autoreactive T cells in autoimmune conditions.Clinicaltrials using T cell vaccination to deplete autoreactive T cells in human autoimmune conditions have begun toreveal the pathologic relevance of various autoimmune T cell populations in the disease processes,providing aunique opportunity to test the autoimmune theories in a clinical setting.Cellular & Molecular Immunology.2004;1(5):321-327.     

Immunoregulatory role of B7-H1 in chronicity of inflammatory responses

Pathogenesis of most chronic human diseases, including chronic infections, autoimmune diseases and cancers, often involves a persistent, unresolved inflammatory response. The molecular mechanisms that determine the conversion of an acute inflammatory response into a chronic process had puzzled researchers for many years. Recent studies reveal that B7-H1 （CD274, PD-L1）, a newly identified co-stimulatory molecule, possesses dual functions of co-stimulation of naive T cells and inhibition of activated effector T cells. The aberrant cellular expression and deregulated function of B7-H1 have been reported during chronic viral and intracellular bacterial infection, as well as in many autoimmune diseases and cancers. Importantly, the deregulation of B7-H1＇s dual functions appears to be associated with a prolonged and incomplete immune response by luring naive T cells for activation and dampening activated effector T cells. Moreover, development of strategies targeting B7-H1 signals provides a new and promising approach to manipulate the devastating diseases associated with chronic inflammation. Thus, B7-H1 may play a critical immunoregulatory role in the chronicity of inflammatory responses. Cellular ＆ Molecular Immunology. 2006;3（3）：179-187.     

Loss of TCR responsiveness during thymic education imprints the ＇innate＇ signature on γδ T cells

T cells expressing γδ determinants are considered innate-like and deviate from the classical αβ T cells in many aspects, including the rapid production of lymphokines following the recognition of ＇naked＇ antigens by the invariant T-cell receptor and failure to differentiate in the periphery.     

The immune response induced by hepatitis B virus principal antigens

Hepatitis B virus （HBV） infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellniar carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.     

禽流感H5N1亚型病毒感染BALB/c小鼠的免疫应答

目的 研究禽流感H5N1病毒感染BALB/c小鼠后对宿主细胞免疫功能和细胞因子水平变化的影响,探讨禽流感H5N1病毒感染哺乳动物的免疫发病机制.方法 选用鹅源禽流感H5N1病毒感染BALB/c小鼠,采用流式细胞仪检测血液和脾脏T淋巴细胞及其亚群的变化,采用ELISA检测血液中细胞岗子(IFN-γ、TNF-α、IL-4、IL-18、IL-10、IL-2)及禽流感H5N1病毒特异性抗体的变化.结果 禽流感H5N1病毒感染可引起对宿主短暂的、可恢复的细胞免疫功能损伤:血液CD3+、CD4+、CD8+ T淋巴细胞数量于染毒后第2～4天下降(第4天为最低值),脾脏T淋巴细胞数最于染毒后第5～8天下降(第6天为最低值),然后均逐渐恢复到正常水平.染毒后血液细胞因子变化表现为:血清IFN-γ、TNF-α水平下降,IL-4、IL-18、IL-10水平上升,IL-2水平无明显变化.从感染第7天开始检测H5N1禽流感特异性抗体为阳性,抗体水平逐渐升高至实验结束的感染第14天.结论 H5N1禽流感病毒感染可引起宿主T细胞免疫功能低下是其主要的免疫病理改变之一,细胞因子表达失平衡或过多的表达都可能对宿主产生免疫病理损伤.

Abstract：

Objective To study the cell immunity and eytokines responses to avian influenza A H5N1 virus infections in a BALB/c model to better understand the pathogenesis of H5N1 avian influenza disease. Methods Two hundred and twenty BALB/c mice of the infected group were inoculated with 0.1 ml (10-4.875 TCID50) of A/Goose/Guangdong/NH/2003 ( H5N1 ) virus intra-nasally. Fifty control mice received noninfectious allantoic fluid and another fifty control mice received normal sodium. Blood and spleen samples were collected from the live mice every 24 h during the 14 d post-infection. The changes of CD3 + T cells , CD4 + T cells, CD8 + T cells for cell immunity in blood circulation and spleen were detected by flow cytometry. And the cytokines and antibody responses in blood circulation were detected by ELISA. Necropsy was performed on mice that died during the experiment and those euthanized at end of study. Results Avian influenza A( H5N1) virus infections can make damages to the cell immune system transiently. The CD3 + T cells, CD4 + T cells, CDS + T cells declined at 24 days post infection in blood circulation and declined at 5-8 days in spleen, then recovered to the normal level gradually. The eytokines responses to the infections can be detected: the level of IFN-γ,TNF-α declined, IL-4, IL-18, IL-10 increased, and IL-2 changed little. The antibody increased rapidly from day 7 post infection until the end of the study (day 14 post infection). Conclusion Collectively, avian influenza A(H5N1) virus can cause cell immunity deficiency and an imbalance in the level of eytokines, which may contribute to the unusual severity of disease caused by the H5N1 avian influenza virus.     

The ex vivo Microenviroments in MLTC of Poorly Immunogenic Tumor Cells Facilitate Polarization of CD4^＋ CD25^＋ Regulatory T Cells

CD4^＋CD25^＋ regulatory T （TR） cells play an important role in maintaining a balanced peripheral immune system. Recent studies have shown that TR cells may also play a key role in suppressing anti-tumor immune response. In order to investigate the tumor immune microenvironment and its influence on TR polarization, poorly immunogenic tumor cell line Ds （C57BL/6, H-2^b）, immunogenic tumor cell lines FBL3 （C57BL/6, H-2^b） and H22 BALB/c, H-2^d） were used to establish the syngeneic/allogeneic, poorly immunogenic/immunogenic mixed lymphocytes-tumor cell culture （MLTC）. Our results revealed that the proportion of CD4^＋CD25^＋ T cells in MLTC of syngeneic primed splenocytes stimulated with D5 tumor cells was higher than that with H22 cells （0.43% vs 0.044%, and the similar results appeared in allogeneic splenocytes stimulated with D5 tumor cells （0.39% vs 0.04%）. The splenocytes stimulated with supernatant from syngeneic MLTC of D5 tumor cells demonstrated higher proportion of CD4^＋CD25^＋ cells than that from allogeneic MLTC of D5 tumor cells, and the splenocytes stimulated with supernatant from syngeneic or allogeneic MLTC of H22 tumor cells generated lower proportion of CD4^＋CD25^＋ T cells than that of D5 tumor cells. The TGF-β1 and Th2-oriented cytokines （IL-4 and IL-10） were dominated in supernatants of syngeneic MLTC of poorly immunogenic tumor cells. Our results provided useful information for studying the mechanisms underlying tumor immune surveillance as well as for the tumor immunotherapy.     

Modulation of HLA expression in human cytomegalovirus immune evasion

Human cytomegalovirus （hCMV） has evolved multiple mechanisms to escape the host immune recognition and innate or adaptive immune responses. Among them, hCMV has developed strategies to modulate the expression and/or function of human leukocyte antigens （HLAs）, including by encoding series of infection stage-dependent hCMV proteins to detain and destroy the expression of HLA molecules on the surface of infected cells. This disturbs the antigen presentation and processing, by encoding MHC class I homologues or selective up-regulation of particular HLA class I molecules binding to NK cell inhibitory receptors, and by encoding specific ligand antagonists to interfere with NK cell activating receptors. Here we discussed the molecular mechanisms utilized by the hCMV to alter the formation, transportation and expression of HLA antigens on the infected cell surface. The knowledge about hCMV modulating HLA expression could benefit us to further understand the pathogenesis of viral diseases and may eventually develop novel effective immunotherapies to counteract viral infections and viral associated diseases.     

The Role of Innate Immune Cells in the Response of Heat-Treated Mycobacterium tuberculosis （M.tb） Antigens Stimulating PBMCs

The proliferation response of γδT cells to the antigen from heat-treated Mycobacterium tuberculosis H37Ra(M.tb Ag)was used as a good model in γδT cell research.From preliminary research it is found that activatedNK cells positively elevated γδT cells proliferation after simulating PBMCs with M.tb Ag.To investigatedifferent behaviors of NK cells,γδNKT cells,γδT cells and relationships between these cell subsets,activationand proliferation of different cell subsets of PBMCs in response to M.tb Ag were analyzed.We demonstratedthat NK cells,γδNKT cells and γδT cells could be activated after stimulation with M.tb Ag.γδNKT cells and γδTcells proliferated while the number of NK cells decreased after 11 day-simulation with M.tb Ag.Meanwhile,atthe early time of stimulation the cytotoxicity of PBMCs was enhanced.Cellular & Molecular Immunology.2004;1(6):467-470.