[show abstract][hide abstract] ABSTRACT: During pathogenic influenza virus infection, robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction all contribute to morbidity and mortality. Earlier we reported that modulation of sphingosine-1-phosphate-1 receptor (S1P1R) signaling provided a chemically tractable approach for the effective blunting of cytokine storm, leading to the improvement of clinical and survival outcomes. Here, we show that S1P1R agonist treatment suppresses global cytokine amplification. Importantly, S1P1R agonist treatment was able to blunt cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways. S1P1R signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways for myeloid differentiation primary response gene 88 (MyD88) and IFN-β promoter stimulator-1 signaling, indicating a common pathway inhibition of cytokine storm. We identify the MyD88 adaptor molecule as responsible for the majority of cytokine amplification observed following influenza virus challenge.
Proceedings of the National Academy of Sciences 02/2014; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Influenza infection of humans remains an important public health problem. Vaccine strategies result in a significant but only partial control (65–85%) of infection. Thus, chemotherapeutic approaches are needed to provide a solution both for vaccine failures and to limit infection in the unvaccinated population. Previously (9 and 10) documented that sphingosine-1-phosphate 1 receptor (S1P1R) agonists significantly protected mice against pathogenic H1N1 influenza virus by limiting immunopathologic damage while allowing host control of the infection. Here we extend that observation by documenting S1P1R agonist can control pathogenic H1N1 influenza infection in ferrets. S1P1R agonist was more effective in reducing pulmonary injury than the antiviral drug oseltamivir but, importantly, combined therapy was significantly more effective than either therapy alone.
Virology 01/2014; s 452–453:152–157. · 3.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: The paramyxovirus, pneumonia virus of mice (PVM), is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8(+) T-cell repertoire in susceptible C57Bl/6 mice. In total, 15 PVM-specific CD8(+) T-cell epitopes restricted by H-2D(b) and/or K(b) were identified. These data open the door for using widely profiled, genetically manipulated, C57Bl/6 mice to study the contribution of epitope-specific CD8(+) T-cells to PVM pathogenesis.
[show abstract][hide abstract] ABSTRACT: Follicular helper T cells (TFH cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised TFH differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell-specific expression of a transgene encoding miR-17∼92 spontaneously accumulated TFH cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4(+) T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in TFH differentiation and establish PHLPP2 as an important mediator of their function in this process.
[show abstract][hide abstract] ABSTRACT: In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP(Sc)) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. While the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP(Sc) in the brain, none of the inoculated heart samples resulted in disease or accumulation of PrP(Sc). Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.
[show abstract][hide abstract] ABSTRACT: Persistent viral infections are the result of a series of connected events that culminate in diminished immunity and the inability to eliminate infection. By building our understanding of how distinct components of the immune system function both individually and collectively in productive versus abortive responses, new potential therapeutic targets can be developed to overcome immune dysfunction and thus fight persistent infections. Using lymphocytic choriomeningitis virus (LCMV) as a model of a persistent virus infection and drawing parallels to persistent human viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), we describe the cellular relationships and interactions that determine the outcome of initial infection and highlight immune targets for therapeutic intervention to prevent or treat persistent infections. Ultimately, these findings will further our understanding of the immunologic basis of persistent viral infection and likely lead to strategies to treat human viral infections.
[show abstract][hide abstract] ABSTRACT: Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generation of virus-specific CD8(+) T cells and clearance of the infection. Inoculation of newborn mice with rLCMV Cl13/LASV Gp resulted in a life-long persistent infection. Interestingly, adoptive transfer of rLCMV Cl13/LASV Gp immune memory cells into such persistently infected mice failed to purge virus but, in contrast, cleared virus from mice persistently infected with wt LCMV Cl13.
[show abstract][hide abstract] ABSTRACT: During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell-dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.
[show abstract][hide abstract] ABSTRACT: Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion's physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion.
[show abstract][hide abstract] ABSTRACT: The sphingosine-1-phosphate (S1P) receptor signaling system has biological and medical importance and is the first lipid G protein-coupled receptor (GPCR) structure to be solved to 2.8-Å resolution. S1P binds to five high-affinity GPCRs generating multiple downstream signals that play essential roles in vascular development and endothelial integrity, control of cardiac rhythm, and routine oral treatment of multiple sclerosis. Genetics, chemistry, and now structural biology have advanced this integrated biochemical system. The S1P receptors have a novel N-terminal fold that occludes access to the binding pocket from the extracellular environment as well as orthosteric and bitopic ligands with very different physicochemical properties. S1P receptors and metabolizing enzymes have been deleted, inducibly deleted, and knocked in as tagged or altered receptors in mice. An array of genetic models allows analysis of integrated receptor function in vivo. We can now directly understand causal relationships among protein expression, signal, and control points in physiology and pathology. Expected final online publication date for the Annual Review of Biochemistry Volume 82 is June 02, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Annual review of biochemistry 03/2013; · 29.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viruses have unique lifestyles. To describe the pathogenesis and significance of viral infection in terms of host responses, resultant injury, and therapy, we focused on two RNA viruses: lymphocytic choriomeningitis (LCMV) and influenza (Flu). Many of the currently established concepts and consequences about viruses and immunologic tolerance, virus-induced immunosuppression, virus-induced autoimmunity, immune complex disease, and virus-lymphocyte and virus-dendritic cell interactions evolved through studies of LCMV in its natural murine host. Similarly, the mechanisms, aftermath, and treatment of persistent RNA viruses emerged, in large part, from research on LCMV. Analysis of acute influenza virus infections uncovered the prominent direct role that cytokine storm plays in the pathogenesis, morbidity, and mortality from this disease. Cytokine storm of influenza virus infection is initiated via a pulmonary endothelial cell amplification loop involving IFN-producing cells and virus-infected pulmonary epithelial cells. Importantly, the cytokine storm is chemically treatable with specific agonist therapy directed to the sphingosphine 1 phosphate receptor 1, which is located on pulmonary endothelial cells, pointing to the endothelial cells as the gatekeepers of this hyperaggressive host immune response.
Proceedings of the National Academy of Sciences 01/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: A recent publication indicated that overexpression of Axl, a cellular receptor that negatively regulates TLR signaling, enhanced entry of viruses pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (LCMV) in vitro. In testing the biological relevance of these observations, we found neither differences in viral kinetics between LCMV-infection of Axl(-/-) and wild-type mice nor in T-cell responses prior to spontaneous viral clearance. Thus, Axl is not required for productive LCMV infection of mice.
[show abstract][hide abstract] ABSTRACT: The cytokine storm is an aggressive immune response characterized by the recruitment of inflammatory leukocytes and exaggerated levels of cytokines and chemokines at the site of infection. Here we review evidence that cytokine storm directly contributes to the morbidity and mortality resulting from influenza virus infection and that sphingosine-1-phosphate (S1P) receptor agonists can abort cytokine storms providing significant protection against pathogenic human influenza viral infections. In experiments using murine models and the human pathogenic 2009 influenza viruses, S1P1 receptor agonist alone reduced deaths from influenza virus by over 80% as compared to lesser protection (50%) offered by the antiviral neuraminidase inhibitor oseltamivir. Optimal protection of 96% was achieved by combined therapy with the S1P1 receptor agonist and oseltamivir. The functional mechanism of S1P receptor agonist(s) action and the predominant role played by pulmonary endothelial cells as amplifiers of cytokine storm during influenza infection are described.
[show abstract][hide abstract] ABSTRACT: Influenza-induced lung edema and inflammation are exacerbated by a positive feedback loop of cytokine and chemokine production termed a 'cytokine storm', a hallmark of increased influenza-related morbidity and mortality. Upon infection, an immune response is rapidly initiated in the lungs and draining lymph node, leading to expansion of virus-specific effector cells. Using two-photon microscopy, we imaged the dynamics of dendritic cells (DC) and virus-specific eGFP(+)CD8(+) T cells in the lungs and draining mediastinal lymph nodes during the first two weeks following influenza infection. Three distinct phases of T cell and CD11c(+) DC behavior were revealed: 1) Priming, facilitated by the arrival of lung DCs in the lymph node and characterized by antigen recognition and expansion of antigen-specific CD8(+) T cells; asymmetric T cell division in contact with DCs was frequently observed. 2) Clearance, during which DCs re-populate the lung and T cells leave the draining lymph node and re-enter the lung tissue where enlarged, motile T cells come into contact with DCs and form long-lived interactions. 3) Maintenance, characterized by T-cell scanning of the lung tissue and dissociation from local antigen presenting cells; the T cells spend less time in association with DCs and migrate rapidly on collagen. A single dose of a sphingosine-1-phosphate receptor agonist, AAL-R, sufficient to suppress influenza-induced cytokine-storm, altered T cell and DC behavior during influenza clearance, delaying T cell division, cellular infiltration in the lung, and suppressing T-DC interactions in the lung. Our results provide a detailed description of T cell and DC choreography and dynamics in the lymph node and the lung during influenza infection. In addition, we suggest that phase lags in T cell and DC dynamics induced by targeting S1P receptors in vivo may attenuate the intensity of the immune response and can be manipulated for therapeutic benefit.
PLoS ONE 01/2013; 8(3):e58033. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.
[show abstract][hide abstract] ABSTRACT: We quantified CD8 T cells needed to cause type 1 diabetes and studied the anatomy of the CD8 T cell/beta (β) cell interaction at the immunologic synapse. We used a transgenic model, in situ tetramer staining to distinguish antigen specific CD8 T cells from total T cells infiltrating islets and a variety of viral mutants selected for functional deletion(s) of various CD8 T cell epitopes. Twenty percent of CD8 T cells in the spleen were specific for all immunodominant and subdominant viral glycoprotein (GP) epitopes. CTLs to the immunodominant LCMV GP33-41 epitope accounted for 63% of the total (12.5% of tetramers). In situ hybridization analysis demonstrated only 1 to 2% of total infiltrating CD8 T cells were specific for GP33 CD8 T cell epitope, yet diabetes occurred in 94% of mice. The immunologic synapse between GP33 CD8 CTL and β cell contained LFA-1 and perforin. Silencing both immunodominant epitopes (GP33, GP276-286) in the infecting virus led to a four-fold reduction in viral specific CD8 CTL responses, negligible lymphocyte infiltration into islets and absence of diabetes.
[show abstract][hide abstract] ABSTRACT: Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.
[show abstract][hide abstract] ABSTRACT: Interleukin-10 (IL-10) is an important factor involved in T-cell dysfunction during persistent viral infection. Although several factors can negatively regulate T-cell activity, targeting of the IL-10 pathway alone is sufficient to regenerate T-cell activity and increase viral control. How IL-10 mediates these effects is unclear. Here, we investigated the cellular source of IL-10 necessary for establishing T-cell exhaustion and viral persistence, using IL-10 reporter mice (VertX), cell-type-specific IL-10 and IL-10 receptor deletion mice, and bone marrow chimeric mice. During establishment of viral persistence, the cellular subset with the most prevalent expression of IL-10 was CD8α(-)CD4(+) dendritic cells (DCs), which produced IL-10 with increasing kinetics until 9 d postinfection. After this time point, DCs exhibited a modest decline in percentage of IL-10(+) cells whereas B cells and CD4(+) T cells increased minimally. Further analysis of the DC population demonstrated that IL-10 was primarily expressed in infected DCs. These DCs were a notable source of IL-10 as mutant mice with a DC-specific deletion of IL-10 had significantly decreased serum levels. Interestingly, viral infection was not directly causative of IL-10 expression; rather, IL-10 production appeared to be linked to type I IFN signaling. Our findings further illuminate the contribution of DCs to the production of IL-10 and to viral persistence.
Proceedings of the National Academy of Sciences 08/2012; 109(35):14116-21. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Copper influences the pathogenesis of prion disease, but whether it is beneficial or detrimental remains controversial. Copper homeostasis is also essential for normal physiology, as highlighted by the spectrum of diseases caused by disruption of the copper transporting enzymes ATP7A and ATP7B. Here, by using a forward genetics approach in mice, we describe the isolation of three alleles of Atp7a, each with different phenotypic consequences. The mildest of the three, Atp7a(brown), was insufficient to cause lethality in hemizygotes or mottling of the coat in heterozygotes, but did lead to coat hypopigmentation and reduced copper content in the brains of hemizygous males. When challenged with Rocky Mountain Laboratory scrapie, the onset of prion disease was delayed in Atp7a(brown) mice, and significantly less proteinase-resistant prion protein was found in the brains of moribund Atp7a(brown) mice compared with WT littermates. Our results establish that ATP7A-mediated copper homeostasis is important for the formation of pathogenic proteinase-resistant prion protein.
Proceedings of the National Academy of Sciences 08/2012; 109(34):13733-8. · 9.74 Impact Factor