Ralph A. Tripp

Pattern recognition receptors TLR4 and CD14 mediate response to respiratorysyncytial virus

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

Defective Lymphoid Development in Mice Lacking Jak3

The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common γ (γc) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in γc signaling and lymphoid development.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

ABSTRACT The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14+ cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections.

The rules for T‐cell‐mediated control of viruses that infect via the respiratory mucosae show both common themes and differences depending on the nature of the pathogen. Virus‐specific CD8+ cytotoxic T lymphocytes (CTLs) are the key effectors of virus clearance in mice infected with both negative strand RNA viruses (influenza and Sendai) and a DNA virus, the murine γ‐herpesvirus68 (MHV‐68). Recently completed experiments establish that these activated CD8+ T cells indeed operate primarily via contact‐dependent lysis, Perform‐mediated cytotoxicity seems to be the preferred mode, though a Fas‐based mechanism can apparently serve as an alternative mechanism. Immune CD4+ T cells functioning in the absence of the CD8+ subset cannot eliminate MHV‐68 from lung epithelial cells, are somewhat less efficient than the CD8+ CTLs at clearing the RNA viruses, and are generally ineffectual in mice that lack B lymphocytes. Though cytokine secretion by CD4+ and CD8+ T cells in the virus‐infected king may promote both T‐cell extravasation and macrophage activation, such processes are not alone sufficient to deal consistently with any d these infections. However, CD4+ T help is mandatory for an effective B‐cell response, and can operate lo promote the clonal expansion of virus‐specific CD8+ T cells in the lymph nodes and spleen. Furthermore, a concurrent CD4+ T‐cell response seems to be essential for maintaining continued CD8+ T‐cell surveillance and effector capacity through the persistent, latent phase of MHV‐68 infection in B cells. Thus, the evidence to date supports a very traditional view: CD8+ T cells function mainly as killers and the CD4+ T cells as helpers in these respiratory virus infections.

CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein.

Chemokines are chemoattractant proteins that are divided into subfamilies based upon cysteine signature motifs termed C, CC, CXC and CX3C. Chemokines have roles in immunity and inflammation that affect cell trafficking and activation of T cells as well as cells of the innate immune system. We report here CX3C chemokine mimicry for the G glycoprotein of respiratory syncytial virus (RSV) and show binding to CX3CR1—the specific receptor for the CX3C chemokine fractalkine—and induction of leukocyte chemotaxis. We also show that CX3CR1 facilitates RSV infection of cells. Thus, G glycoprotein interaction with CX3CR1 probably plays a key role in the biology of RSV infection.

Forced degradation of Fas inhibits apoptosis in adenovirus-infected cells

DNA viruses have evolved elaborate mechanisms to overcome host antiviral defences. In adenovirus-infected cells, programmed cell death (apoptosis) induced by the cytokine tumour necrosis factor (TNF) is inhibited by several adenovirus-encoded proteins. Occupation of the cell-surface receptor Fas, a member of the TNF-receptor superfamily that is expressed on most cell types, triggers apoptosis of that cell. Here we show that the adenovirus RID (for receptor internalization and degradation) protein complex, which is an inhibitor of TNF-induced apoptosis, mediates internalization of cell-surface Fas and its destruction inside lysosomes within the cell. Fas has not previously been shown to be internalized and then degraded. RID also mediates internalization of the receptor for epidermal growth factor,, but it does not affect the transferrin receptor or class I antigens of the major histocompatibility complex. Removal of Fas from the surface of adenovirus-infected cells expressing RID may allow infected cells to resist Fas-mediated cell death and thus promote their survival.

Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2

ABSTRACT Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.

Rapid microRNA (miRNA) detection and classification via surface-enhanced Raman spectroscopy (SERS)

microRNAs (miRNA) are recognized as regulators of gene expression during development and cell differentiation as well as biomarkers of disease. Development of rapid and sensitive miRNA profiling methods is essential for evaluating the pattern of miRNA expression that varies across normal and diseased states. The ability to identify miRNA expression patterns is limited to cumbersome assays that often lack sensitivity and specificity to distinguish between different miRNA families and members. We evaluated a surface-enhanced Raman scattering (SERS) platform for detection and classification of miRNAs. The strength of the SERS-based sensor is its sensitivity to detect extremely low levels of analyte and specificity to provide the molecular fingerprint of the analyte. We show that the SERS spectra of related and unrelated miRNAs can be detected in near-real time, that detection is sequence dependent, and that SERS spectra can be used to classify miRNA patterns with high accuracy.

Cortisol mediated suppression of salmonid lymphocyte responses invitro

The suppressive activity of cortisol on the in vitro induction of coho salmon (Oncorhynchus kisutch) B cell activation was examined. Suppression was observed with splenic and pronephric (anterior kidney) derived lymphocytes. The kinetics of cortisol-induced suppression revealed distinct differences in the sensitivity of splenic and pronephric lymphocytes. Pronephric lymphocytes were only sensitive to cortisol early in the induction of the antibody response, whereas the splenic cells were sensitive to cortisol throughout the culture period. Addition of supernatants from antigen stimulated pronephric cultures completely restored the ability of pronephric lymphocytes to produce an antibody response, suggesting that this glucocorticoid-suppression may be mediated by inhibition of lymphokine production.

RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy

ABSTRACT We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.