R.S. Peebles

Suppression of airway hyperresponsiveness induced by ovalbumin sensitisation and RSV infection with Y-27632, a Rho kinase inhibitor

Background: Smooth muscle contraction is one of the hallmarks of asthma. A recently developed pyridine derivative, Y-27632, a selective Rho kinase inhibitor, has been reported to inhibit the smooth muscle contraction of human and animal trachea in ex vivo systems but its effect in animal models of airway hyperresponsiveness (AHR) has not been examined. The purpose of this study was to evaluate the effect of Y-27632 in a murine model of allergic and virally induced AHR. Methods: Baseline lung resistance and methacholine induced AHR were measured in mice sensitised to ovalbumin (OVA) and also in mice infected with respiratory syncytial virus (RSV) following ovalbumin sensitisation (OVA/RSV). Results: Time course and dose ranging experiments indicated that 30 mg/kg Y-27632 given by gavage 2 hours before methacholine challenge significantly reduced baseline lung resistance and prevented AHR in OVA sensitised mice. Y-27632 also suppressed AHR induced by the bronchospastic agent serotonin in OVA sensitised mice and prevented methacholine induced AHR in OVA/RSV mice. Conclusions: These results suggest that the signalling pathway mediated through Rho kinase may have an important role in bronchial smooth muscle tone in allergen induced and virus induced AHR and should be considered as a novel target for asthma treatment.

Prostaglandin I2 Signaling and Inhibition of Group 2 Innate Lymphoid Cell Responses

RATIONALE Group 2 innate lymphoid cells (ILC2s) robustly produce IL-5 and IL-13, cytokines central to the asthma phenotype; however, the effect of prostaglandin (PG) I2 on ILC2 function is unknown. OBJECTIVES To determine the effect of PGI2 on mouse and human ILC2 cytokine expression in vitro and the effect of endogenous PGI2 and the PGI2 analog cicaprost on lung ILC2s in vivo. METHODS Flow-sorted bone marrow ILC2s of wild-type (WT) and PGI2 receptor-deficient (IP(-/-)) mice were cultured with IL-33 and treated with the PGI2 analog cicaprost. WT and IP(-/-) mice were challenged intranasally with Alternaria alternata extract for 4 consecutive days to induce ILC2 responses, and these were quantified. Prior to A. alternata extract, challenged WT mice were treated with cicaprost. Human flow-sorted peripheral blood ILC2s were cultured with IL-33 and IL-2 and treated with the PGI2 analog cicaprost. MEASUREMENT AND MAIN RESULTS We demonstrate that PGI2 inhibits IL-5 and IL-13 protein expression by IL-33-stimulated ILC2s purified from mouse bone marrow in a manner that was dependent on signaling through the PGI2 receptor IP. In a mouse model of 4 consecutive days of airway challenge with an extract of A. alternata, a fungal aeroallergen associated with severe asthma exacerbations, endogenous PGI2 signaling significantly inhibited lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5- and IL-13-expressing ILC2s, as well as the mean fluorescence intensity of IL-5 and IL-13 staining. In addition, exogenous administration of a PGI2 analog inhibited Alternaria extract-induced lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5- and IL-13-expressing ILC2s and the mean fluorescence intensity of IL-5 and IL-13 staining. Finally, a PGI2 analog inhibited IL-5 and IL-13 expression by human ILC2s that were stimulated with IL-2 and IL-33. CONCLUSIONS These results suggest that PGI2 may be a potential therapy to reduce the ILC2 response to protease-containing aeroallergens, such as Alternaria.

The role of prostaglandins in allergic lung inflammation and asthma.

Prostaglandins (PGs) are products of the COX pathway of arachidonic acid metabolism. There are five primary PGs, PGD2, PGE2, PGF2, PGI2 and thromboxane A2, all of which signal through distinct seven transmembrane, G-protein coupled receptors. Some PGs may counteract the actions of others, or even the same PG may have opposing physiologic or immunologic effects, depending on the specific receptor through which it signals. In this review, we examine the effects of COX activity and the various PGs on allergic airway inflammation and physiology that is associated with asthma. We also highlight the potential therapeutic benefit of targeting PGs in allergic lung inflammation and asthma based on basic science, animal model and human studies.

RSV replication is attenuated by counteracting expression of the suppressor of cytokine signaling (SOCS) molecules.

Human RSV causes an annual epidemic of respiratory tract illness in infants and in elderly. Mechanisms by which RSV antagonizes IFN-mediated antiviral responses include inhibition of type I IFN mRNA transcription and blocking signal transduction of JAK/STAT family members. The suppressor of cytokines signaling (SOCS) gene family utilizes a feedback loop to inhibit cytokine responses and block the activation of the JAK/STAT signaling pathway. To evaluate the potential of SOCS molecules to subvert the innate immune response to RSV infection, eight SOCS family genes were examined. RSV infection up-regulated SOCS1, SOCS3, and CIS mRNA expression in HEp-2 cells. Suppression of SOCS1, SOCS3 and CIS by short interfering ribonucleic acid (siRNA) inhibited viral replication. Furthermore, inhibition of SOCS1, SOCS3, or CIS activated type I IFN signaling by inducing STAT1/2 phosphorylation. These results suggest that RSV infection escapes the innate antiviral response by inducing SOCS1, SOCS3 or CIS expression in epithelial cells.

Bugs and Asthma: A Different Disease?

The prevalence of asthma has dramatically increased in recent decades. Exacerbations of asthma are a large contributor to asthma-related costs, and are primarily caused by viral and atypical bacterial infections. Rhinoviruses (RVs) are the most common viruses detected after an asthma exacerbation. RVs, respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) viral infections early in life can induce wheezing and are associated with the development of asthma later in life. Atypical bacterial infections from Mycoplasma pneumoniae and Chlamydia pneumoniae have also been linked to chronic asthma and potential asthma exacerbations. In this article, we will discuss recent developments in viral infections, specifically RV, RSV, and hMPV, and atypical bacterial infections as causes of asthma exacerbations, including new data focusing on the host immune response in airway epithelial cells and animal models of infection.

Respiratory viruses and asthma.

Viral infections have become increasingly recognized as a significant cause of asthma exacerbations, mainly because of improved viral detection techniques. Unfortunately, the ability to specifically treat viral infections and to limit the asthma morbidity associated with these agents has not kept pace with diagnostic technology. This article focuses on current concepts of the epidemiology of viruses in asthma exacerbations, investigations studying the physiologic and immunologic consequences of viral infection, and potential therapies to minimize virally-induced airway hyperresponsiveness. To impact this significant health problem, researchers must definitively ascertain the mechanisms by which viruses induce airway reactivity and must develop rational, safe approaches to prevent the consequences of viral infection in the patient with asthma.

Functional polymorphism of the promoter region of the prostacyclin synthase gene and severity of RSV infection in hospitalized children

Prostaglandin I2 (PGI2) protects against RSV‐induced illness in mice. A variable‐number tandem repeat (VNTR) polymorphism has been detected in the promoter region of the PGI2 synthase (PGIS) gene. We sought to determine if PGI2 concentrations or polymorphisms of the PGIS gene correlate with severity of RSV lower respiratory tract infections (LRTI) in human infants. VNTR polymorphisms were studied in 81 previously healthy children between birth and 12 months of age who were hospitalized for LRTI due to RSV and 98 healthy adult control subjects. The severity of RSV infection was quantified using a clinical scoring system, and infant urine samples were collected during the acute illness for measurement of the urinary metabolite of PGI2. There were no significant differences in the overall distribution of alleles and genotypes between infants with RSV LRTI and the control subjects. The severity of RSV infection significantly inversely correlated with urinary PGI2 metabolite concentrations. The urinary PGI2 metabolite concentration correlated with the number of VNTR. The presence of a genotype with a low number VNTR repeats significantly correlated with the most severe RSV LRTI, and genotypes with the highest number of VNTR correlated with the least severe RSV LRTI. A functional polymorphism in the promoter region of the PGIS gene is associated with both significant differences in urinary PGI2 concentrations during RSV LRTI, and severity of RSV infection in previously healthy infants. J. Med. Virol. 80:2015–2022, 2008.

DSCG reduces RSV-induced illness in RSV-infected mice.

Respiratory syncytial virus (RSV) is one of the pathogens generally associated with the common cold, lower respiratory infection, and exacerbation of asthma. Disodium cromoglycate (DSCG) is a safe and widely used drug for the prevention of bronchial asthma and allergic rhinitis attacks. The effect of DSCG on acute upper respiratory tract viral infections remains controversial. The purpose of the study was to investigate the effects of DSCG on parameters of RSV induced‐illness. Using a well‐characterized murine model of RSV infection, the effect of DSCG on RSV‐induced illness was evaluated by body weight, respiratory function, viral replication, level of IFN‐γ in lungs, serology, and histopathology. Mice treated with DSCG were protected against RSV‐induced weight loss. The baseline Penh in RSV‐infected mice treated with DSCG was less than that in mice treated with saline. In methacholine challenge, the increase in Penh in RSV‐infected mice treated with DSCG was suppressed to the same level as that in the mock‐infected group. Further, there were no differences in viral replication between the mice treated with DSCG and those treated with saline, and the level of inflammation observed in the lungs in RSV‐infected mice treated with DSCG was not as severe as that in mice treated with saline. These findings indicate that DSCG may be an effective agent for the prevention of RSV induced disease and the relief of symptoms of RSV infection. J. Med. Virol. 81:354–361, 2009.

STAT4 Deficiency Fails To Induce Lung Th2 or Th17 Immunity following Primary or Secondary Respiratory Syncytial Virus (RSV) Challenge but Enhances the Lung RSV-Specific CD8+ T Cell Immune Response to Secondary Challenge

ABSTRACT Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4−/− mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4−/− mice and used STAT1−/− mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4−/− mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4−/− mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4−/− mice compared to WT mice. Following secondary challenge, WT and STAT4−/− mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4−/− mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge. IMPORTANCE STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections.

The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 μm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.