Timur O. Yarovinsky

Respiratory Syncytial Virus Up-regulates TLR4 and Sensitizes Airway Epithelial Cells to Endotoxin

Airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide (LPS)) exposure under normal conditions. This study demonstrates that respiratory syncytial virus (RSV) infection results in increased sensitivity to this environmental exposure. Infection with RSV results in increased expression of Toll-like receptor (TLR) 4 mRNA, protein, and increased TLR4 membrane localization. This permits significantly enhanced LPS binding to the epithelial monolayer that is blocked by disruption of the Golgi. The increased TLR4 results in an LPS-induced inflammatory response as demonstrated by increased mitogen-activated protein (MAP) kinase activity, IL-8 production, and tumor necrosis factor α production. RSV infection also allowed for tumor necrosis factor α production subsequent to TLR4 cross-linking with an immobilized antibody. These data suggest that RSV infection sensitizes airway epithelium to a subsequent environmental exposure (LPS) by altered expression and membrane localization of TLR4. The increased interaction between airway epithelial cells and LPS has the potential to profoundly alter airway inflammation.

Respiratory Syncytial Virus Induces TLR3 Protein and Protein Kinase R, Leading to Increased Double-Stranded RNA Responsiveness in Airway Epithelial Cells

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-κB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.

Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon response with expression of interferon-stimulated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toll-like receptor 7 (TLR7) and TLR9. The fusion-dependent response was dependent on the stimulator of interferon genes STING but was independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant-cell formation.

Respiratory Syncytial Virus Decreases p53 Protein to Prolong Survival of Airway Epithelial Cells

Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1–6 h after infection resulting in a decrease in p53 6–24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.

VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling

Rationale There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries. Objective We sought to address the role of VEGF blockade on remodeling of human arteries in vivo. Methods and Results We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell–induced but not interferon-&ggr;–induced neointimal formation. This inhibitory effect was associated with a reduction in graft T-cell accumulation without affecting T-cell activation. VEGF enhanced T-cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 rather than endothelial cells was used to capture T cells. A subpopulation of CD3+ T cells expressed VEGF receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T-cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells. Conclusions VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.

Respiratory Syncytial Virus Synergizes with Th2 Cytokines to Induce Optimal Levels of TARC/CCL17

Respiratory syncytial virus (RSV) is a ubiquitous virus that preferentially infects airway epithelial cells, causing asthma exacerbations and severe disease in immunocompromised hosts. Acute RSV infection induces inflammation in the lung. Thymus- and activation-regulated chemokine (TARC) recruits Th2 cells to sites of inflammation. We found that acute RSV infection of BALB/c mice increased TARC production in the lung. Immunization of BALB/c mice with individual RSV proteins can lead to the development of Th1- or Th2-biased T cell responses in the lung after RSV infection. We primed animals with a recombinant vaccinia virus expressing either the RSV fusion (F) protein or the RSV attachment (G) protein, inducing Th1- and Th2-biased pulmonary memory T cell responses, respectively. After RSV infection, TARC production significantly increased in the vaccinia virus G-primed animals only. These data suggest a positive feedback loop for TARC production between RSV infection and Th2 cytokines. RSV-infected lung epithelial cells cultured with IL-4 or IL-13 demonstrated a marked increase in the production of TARC. The synergistic effect of RSV and IL-4/IL-13 on TARC production reflected differential induction of NFκB and STAT6 by the two stimuli (both are in the TARC promoter). These findings demonstrate that RSV induces a chemokine TARC that has the potential to recruit Th2 cells to the lung.

Early exposure to IL-4 stabilizes IL-4 mRNA in CD4+ T cells via RNA-binding protein HuR.

The mechanisms regulating IL-4 mRNA stability in differentiated T cells are not known. We found that early exposure of CD4+ T cells to endogenous IL-4 increased IL-4 mRNA stability. This effect of IL-4 was mediated by the RNA-binding protein HuR. IL-4 mRNA interacted with HuR and the dominant binding site was shown within the coding region of IL-4 mRNA. Exposure of CD4+ T cells to IL-4 had no effects on HuR expression or subcellular localization, but triggered HuR binding to IL-4 mRNA. Thus, IL-4 plays a positive role in maintaining IL-4 mRNA stability in CD4+ T cells via a HuR-mediated mechanism.

Macrophage β2 Integrin–Mediated, HuR-Dependent Stabilization of Angiogenic Factor–Encoding mRNAs in Inflammatory Angiogenesis

HuR is a member of the Drosophila Elav protein family that binds mRNA degradation sequences and prevents RNase-mediated degradation. Such HuR-mediated mRNA stabilization, which is stimulated by integrin engagement and is controlled at the level of HuR nuclear export, is critically involved in T-cell cytokine production. However, HuRs role in macrophage soluble factor production, in particular in response to angiogenic stimuli, has not yet been established. We show that the labile transcripts that encode vascular endothelial growth factor and matrix metalloproteinase-9 are stabilized when murine macrophages adhere to the β(2) integrin ligand intercellular adhesion molecule-1. This mRNA stabilization response was absent in bone marrow-derived macrophages obtained from conditional macrophage-specific HuR knockout mice. The microvascular angiogenic response to an inflammatory stimulus (ie, subcutaneous polyvinyl alcohol sponge implantation) was markedly diminished in these macrophage HuR knockout mice despite the equal levels of macrophage localization to those observed in littermate wild-type controls. Furthermore, blood flow recovery and ischemic muscle neovascularization after femoral artery ligation were impaired in the conditional macrophage-specific HuR knockout mice. These results demonstrate that dynamic effects on mRNA, mediated by the RNA-binding and RNA-stabilizing protein HuR, are required for macrophage production of angiogenic factors, which play critical roles in the neovascular responses to a variety of stimuli, including tissue ischemia.

First-Generation Adenovirus Vectors Shorten Survival Time in a Murine Model of Sepsis

Adverse immunological reactions to adenoviral vectors have significantly impacted the utility of this virus for treating genetic and environmentally induced diseases. In this study, we evaluate the effect of adenoviral vectors on an animal model of sepsis. Systemic delivery of first-generation adenoviral vectors to septic mice (cecal ligation and puncture) resulted in a shortened survival time. This effect was not observed with second-generation or inactivated first-generation vectors. The accelerated death was accompanied by a number of important changes in the disease. These changes included increased liver cell apoptosis (including Kupffer cells) and a marked increase in liver bacterial load. In the lung, the combination induced an increase in bacterial load, as well as greater lung injury. In the serum, the combination was associated with decreased TNF-α levels and an increase in bacterial load. Finally, a profound degree of lymphocyte apoptosis was observed in these animals. These observations suggest that prior exposure to first-generation adenovirus gene therapy vectors may worsen the outcome of some forms of sepsis.

A New Mechanism for Inhalational Priming: IL-4 Bypasses Innate Immune Signals

Signaling via innate immune mechanisms is considered pivotal for T cell-mediated responses to inhaled Ags. Furthermore, Th2 cells specific for one inhaled Ag can facilitate priming of naive T cells to unrelated new inhaled Ags, a process we call “Th2 collateral priming”. Interestingly, our previous studies showed that collateral priming is independent of signals via the innate immune system but depends on IL-4 secretion by CD4+ T cells. We thus hypothesized that IL-4 can bypass the need for signals via the innate immune system, considered essential for pulmonary priming. Indeed, we were able to show that IL-4 bypasses the requirement for TLR4- and MyD88-mediated signaling for responses to new allergens. Furthermore, we characterized the mechanisms by which IL-4 primes for new inhaled allergens: “IL-4-dependent pulmonary priming” relies on IL-4 receptor expression on hematopoietic cells and structural cells. Transfer experiments indicate that within the hematopoietic compartment both T cells and dendritic cells need to express the IL-4 receptor. Finally, we were able to show that IL-4 induces recruitment and maturation of myeloid dendritic cells in vivo and increases T cell recruitment to the draining lymph nodes. Our findings bring new mechanistic knowledge to the phenomenon of polysensitization and primary sensitization in asthma.