Simulation and Prediction of the Adaptive Immune Response to Influenza A Virus Infection

The cellular immune response to primary influenza virus infection is complex, involving multiple cell types and anatomical compartments, and is difficult to measure directly. Here we develop a two-compartment model that quantifies the interplay between viral replication and adaptive immunity. The fidelity of the model is demonstrated by accurately confirming the role of CD4 help for antibody persistence and the consequences of immune depletion experiments. The model predicts that drugs to limit viral infection and/or production must be administered within 2 days of infection, with a benefit of combination therapy when administered early, and cytotoxic CD8 T cells in the lung are as effective for viral clearance as neutralizing antibodies when present at the time of challenge. The model can be used to investigate explicit biological scenarios and generate experimentally testable hypotheses. For example, when the adaptive response depends on cellular immune cell priming, regulation of antigen presentation has greater influence on the kinetics of viral clearance than the efficiency of virus neutralization or cellular cytotoxicity. These findings suggest that the modulation of antigen presentation or the number of lung resident cytotoxic cells and the combination drug intervention are strategies to combat highly virulent influenza viruses. We further compared alternative model structures, for example, B-cell activation directly by the virus versus that through professional antigen-presenting cells or dendritic cell licensing of CD8 T cells.Understanding how the immune system combats influenza virus infection and how the virus can affect the immune system is crucial to predicting and designing prophylactic and therapeutic strategies against the infection (58). Antigenic shift and antigenic drift alter the degree to which preexisting immunity can control the virus. These factors also influence whether different arms of the adaptive immune system can cross-react against new strains of the virus. For example, shifts of the hemagglutinin (HA) and neuraminidase (NA) protein sequences limit the ability of antibodies to neutralize new variants of the virus and may make cross-reactive T-cell responses to conserved viral proteins more important. Other viral proteins, such as NS1, affect both the induction of type I interferon as well as the susceptibility of infected cells to interferon-mediated inhibition of viral gene expression (43). The efficiencies of viral replication and cell-to-cell viral spread are altered by mutations in the viral matrix and polymerase genes, while the survival of infected cells can be altered by the viral PB1-F2 protein. These attributes are influenced by mutations in the viral matrix (50, 51) and polymerase (30, 69) genes, while the survival of infected cells can be altered by the viral PB1-F2 protein (17). The multigenic aspect of influenza virus pathogenesis makes experimental prediction difficult and time-consuming. Computer simulation tools would be useful to independently dissect the potential contribution and relative importance of each factor or to investigate unexpected scenarios that are difficult to replicate experimentally.Mathematical models and computer simulations have been widely used to study viral dynamics and immune responses to viral infections, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses (SIV), lymphocytic choriomeningitis virus (19, 55, 60, 61), and influenza A virus (3, 7, 8, 13, 34, 35, 52). More complex compartmental models of the immune system (4, 23) and models incorporating differential delay equations (21, 48, 68) have been used to better reflect the time that cells reside in a particular compartment or the duration of transit between compartments. In this study, we sought to develop a two-compartment mathematical model to assess the individual contributions of antigen presentation and activation of naïve T and B cells by antigen-presenting cells (APC), CD4 T-cell help, CD8 T-cell-mediated cytotoxicity, B cells, and antibody to control influenza A virus (IAV) infection and to explore the influence of anatomical location. We developed a model which represented published experimental findings on primary influenza virus infection. More importantly, the model was used to explore alternative structures for interactions between virus and immune cells, for example, comparing virus kinetics when antigen delivery and immune cell priming occurred through direct interaction of virus and immune cells or through a cellular intermediate. The model predicts that, under some circumstances, changes affecting antigen presentation more strongly impacted viral kinetics than other viral or immune factors (28, 73, 75, 78). This model highlights the importance of the assumptions used to synthesize a model and gaps in our understanding of the immune response regulating primary influenza virus infection. We discuss the implications of these findings for future influenza virus research and theories of influenza virus virulence based on influenza virus-immune system interactions.     

The Transcriptional Response of Drosophila melanogaster to Infection with the Sigma Virus (Rhabdoviridae)

Background

Bacterial and fungal infections induce a potent immune response in Drosophila melanogaster, but it is unclear whether viral infections induce an antiviral immune response. Using microarrays, we examined the changes in gene expression in Drosophila that occur in response to infection with the sigma virus, a negative-stranded RNA virus (Rhabdoviridae) that occurs in wild populations of D. melanogaster.

Principal Findings

We detected many changes in gene expression in infected flies, but found no evidence for the activation of the Toll, IMD or Jak-STAT pathways, which control immune responses against bacteria and fungi. We identified a number of functional categories of genes, including serine proteases, ribosomal proteins and chorion proteins that were overrepresented among the differentially expressed genes. We also found that the sigma virus alters the expression of many more genes in males than in females.

Conclusions

These data suggest that either Drosophila do not mount an immune response against the sigma virus, or that the immune response is not controlled by known immune pathways. If the latter is true, the genes that we identified as differentially expressed after infection are promising candidates for controlling the host''s response to the sigma virus.     

植物对非生物逆境响应的转录调控和代谢谱分析的研究进展

非生物逆境严重影响植物的生长发育,植物响应非生物逆境是通过复杂的转录调控网络和代谢网络实现的。植物转录组学和代谢组学的技术方法有助于研究植物对非生物逆境的应答机制。本文对植物非生物逆境响应中的转录调控和代谢谱分析的研究进展进行了综述。     

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods – an in vitro T cell priming assay and an intracellular cytokine staining (ICS) – were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4⁺ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4⁺ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.     

Transient Transcriptional Regulation of the CYS-C1 Gene and Cyanide Accumulation upon Pathogen Infection in the Plant Immune Response

Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme β-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression. We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic acid-dependent signaling pathway of the plant immune system.The gaseous hormone ethylene is known to regulate multiple physiological and developmental processes in plants, such as seedling emergence, leaf and flower senescence, climacteric fruit ripening, and organ abscission. Ethylene is also involved in the response of plants to abiotic and biotic stresses (Wang et al., 2002; Broekaert et al., 2006; van Loon et al., 2006). Enhanced ethylene production is an early, active response of plants to the perception of pathogen attack and is associated with the induction of defense reactions. During ethylene biosynthesis, S-adenosyl-l-Met is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. ACC is finally oxidized by ACC oxidase to form ethylene, carbon dioxide, and cyanide (Hartley et al., 1998; Wang et al., 2002). Hydrogen cyanide is a colorless and highly volatile liquid. The anion cyanide is toxic and renders the cells of an organism unable to use oxygen, primarily through the chelation of divalent and trivalent metal ions in the prosthetic groups of several metalloenzymes, including copper/zinc superoxide dismutase, catalase, nitrate and nitrite reductase, nitrogenase, peroxidases, and the mitochondrial cytochrome c oxidase (Isom and Way, 1984; Donato et al., 2007).Cyanide must be rapidly detoxified and metabolized by the plant to keep the concentration below toxic levels. Plants detoxify cyanide primarily through the enzyme β-cyanoalanine synthase (CAS), for which considerable levels of activity are constitutively found in many plant species. Rhodanese and mercaptopyruvate sulfurtransferase activities also make minor contributions to the cyanide detoxification process (Miller and Conn, 1980). CAS is a pyridoxal phosphate-dependent enzyme that converts Cys and cyanide to hydrogen sulfide and β-cyanoalanine, which is later converted to Asn, Asp, and ammonia by NIT4 class nitrilases (Piotrowski, 2008). Arabidopsis (Arabidopsis thaliana) plants carry the mitochondrial CAS CYS-C1 (At3g61440; Watanabe et al., 2008), which belongs to the family of β-substituted Ala synthase enzymes. The family also includes the three major O-acetyl-serine(thiol)lyase enzymes OAS-A1 (At4g14880), OAS-B (At2g43750), and OAS-C (At3g59760; Watanabe et al., 2008), the l-Cys desulfhydrase DES1 (At5g28030; Álvarez et al., 2010), the S-sulfocysteine synthase CS26 (At3g03630; Bermúdez et al., 2010), and the functionally unknown cytosolic isoforms CYS-D1 (At3g04940) and CYS-D2 (At5g28020). Mutations in CYS-C1 result in plants that accumulate cyanide and that display abnormal root hair (García et al., 2010), suggesting that cyanide has a signaling role in root development. The lack of the mitochondrial O-acetyl-serine(thiol)lyase isoform OAS-C, which is necessary to detoxify the sulfide released by the CAS activity, causes an accumulation of sulfide and cyanide and a root phenotype similar to the cys-c1 loss-of-function mutant (Álvarez et al., 2012b).Several authors have suggested that cyanide could act as a regulator of other metabolic processes in addition to performing the described role in plant root development (Siegien and Bogatek, 2006). It has been observed that this molecule is released during seed germination and that exogenously applied hydrogen cyanide breaks seed dormancy in several plants (Cohn and Hughes, 1986; Fol et al., 1989; Bogatek et al., 1991; Bethke et al., 2006). The role of cyanide as a regulatory molecule is not restricted to plants, and it has been demonstrated that cyanide is generated in leukocytes from Gly via a peroxidase (Stelmaszyńska, 1986) as well as in the central nervous system, where it has been hypothesized to act as a neuromodulator (Gunasekar et al., 2000; Cipollone and Visca, 2007). Cyanide production can be stimulated by opiates and decreased by treatment with muscarinic receptor agonists (Borowitz et al., 1997; Gunasekar et al., 2004).Despite the variety of known functions for cyanide in different organisms, the role of cyanide production in plants seems to have been unevaluated to date. In cyanogenic plants, cyanide is produced during the degradation of cyanogenic lipids and from the catabolism of cyanogenic glycosides (Poulton, 1990). Cyanide and cyanogenic compounds play an important role in plant defense against herbivores (Zagrobelny et al., 2008). In noncyanogenic plants, cyanide is a coproduct of ethylene biosynthesis. The molecule is also produced during the biosynthesis of camalexin, a phytoalexin formed in Arabidopsis plants upon infection by a large variety of microorganisms, including bacteria, fungi, and oomycetes (Glawischnig, 2007). During camalexin biosynthesis, the Trp-derived intermediate indole-3-acetonitrile is conjugated with Cys and serves as a substrate for the cytochrome P450 enzyme CYP71B15. This enzyme catalyzes the formation of the thiazoline ring as well as the release of cyanide and subsequent oxidative decarboxylation of dihydrocamalexic acid to camalexin (Glawischnig, 2007; Böttcher et al., 2009). Since both cyanide sources, camalexin and ethylene, are produced after pathogen attack, cyanide should be produced at significant levels during the plant response to pathogens. It has been shown that exogenous cyanide can enhance the resistance of tobacco (Nicotiana tabacum) and Arabidopsis leaves to Tobacco mosaic virus and Turnip vein clearing virus, respectively (Chivasa and Carr, 1998; Wong et al., 2002). Recently, it has been demonstrated that exogenously applied cyanide increases the resistance of young rice (Oryza sativa) plants to blast fungus infection, suggesting that cyanide rather than ethylene contributes to plant resistance (Seo et al., 2011).This work aims to further investigate the role of endogenously produced cyanide in the plant immune response by analyzing the behavior of Arabidopsis knockout mutants of the mitochondrial CAS CYS-C1 and the regulation of CYS-C1 in response to pathogen attack.     

Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachochytrium dendrobatidis

Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.     

Chronic Joint Disease Caused by Persistent Chikungunya Virus Infection Is Controlled by the Adaptive Immune Response

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that causes incapacitating disease in humans characterized by intense joint pain that can persist for weeks, months, or even years. Although there is some evidence of persistent CHIKV infection in humans suffering from chronic rheumatologic disease symptoms, little is known about chronic disease pathogenesis, and no specific therapies exist for acute or chronic CHIKV disease. To investigate mechanisms of chronic CHIKV-induced disease, we utilized a mouse model and defined the duration of CHIKV infection in tissues and the associated histopathological changes. Although CHIKV RNA was readily detectable in a variety of tissues very early after infection, CHIKV RNA persisted specifically in joint-associated tissues for at least 16 weeks. Inoculation of Rag1^−/− mice, which lack T and B cells, resulted in higher viral levels in a variety of tissues, suggesting that adaptive immunity controls the tissue specificity and persistence of CHIKV infection. The presence of CHIKV RNA in tissues of wild-type and Rag1^−/− mice was associated with histopathological evidence of synovitis, arthritis, and tendonitis; thus, CHIKV-induced persistent arthritis is not mediated primarily by adaptive immune responses. Finally, we show that prophylactic administration of CHIKV-specific monoclonal antibodies prevented the establishment of CHIKV persistence, whereas therapeutic administration had tissue-specific efficacy. These findings suggest that chronic musculoskeletal tissue pathology is caused by persistent CHIKV infection and controlled by adaptive immune responses. Our results have significant implications for the development of strategies to mitigate the disease burden associated with CHIKV infection in humans.     

肝脏固有免疫系统对肠源性感染的免疫应答与免疫耐受机制

肝特殊的解剖结构及生理特征使其成为暴露肠源性抗原的主要器官。由于肝具有独特的固有免疫系统,在正常情况下,肝分布多种致耐受的抗原提呈细胞,对持续性表达或递呈于肝的肠源性抗原物质,诱发针对该抗原的系统性免疫耐受,避免肝受到不必要的免疫损伤。当炎症发生及肝脏固有免疫系统活化时,则通过免疫效应细胞及免疫效应因子对肠源性病原体发挥强烈地免疫应答以控制感染。该过程形成机制的研究对肝功能的理解及肝性疾病的预防与治疗至关重要。本文就肝固有免疫系统对肠源性感染的免疫应答与免疫耐受形成机制作一综述。     

干扰素刺激基因表达对Peg-IFN抗乙型肝炎病毒免疫应答的影响

为了揭示干扰素刺激基因(interferon-stimulated gene, ISG)表达的改变对乙型肝炎病毒感染治疗效果的影响,本研究检测了干扰素刺激基因STAT1、MX和SOCS3在慢性乙型肝炎患者的外周血单核细胞(peripheral blood mononuclear cell, PBMC)和肝脏样品中的表达情况。结果显示,采用聚乙二醇干扰素(Peg-IFN)治疗后,Peg-IFN应答者的PBMC和肝组织中的STAT1和MX表达水平显著升高,而非应答者的SOCS3表达显著升高。在应答者的活组织检查中,Peg-IFN治疗24 h后细胞核中磷酸化STAT1的染色比例显著增加,而非应答者在治疗前肝细胞核染色比例较高,治疗后染色比例显著减少。此外,治疗前非应答者的肝SOCS3表达水平显著高于应答者,并且随着IFN的治疗SOCS3表达继续增加。本研究表明,STAT1和MX是Peg-IFN抗病毒免疫应答的正向调节因子,而SOCS3 (JAK/STAT途径的负调节因子)激活干扰素刺激基因的负调控,并抑制Peg-IFN的免疫应答。