Jyothi Nayar

Prevention of cold-associated acute inflammation in familial cold autoinflammatory syndrome by interleukin-1 receptor antagonist

BACKGROUND Familial cold autoinflammatory syndrome (FCAS) is an autosomal dominant disorder characterised by recurrent episodes of rash, arthralgia, and fever after cold exposure. The genetic basis of this disease has been elucidated. Cryopyrin, the protein that is altered in FCAS, is one of the adaptor proteins that activate caspase 1, resulting in release of interleukin 1. METHODS An experimental cold challenge protocol was developed to study the acute inflammatory mechanisms occurring after a general cold exposure in FCAS patients and to investigate the effects of pretreatment with an antagonist of interleukin 1 receptor (IL-1Ra). ELISA, real-time PCR, and immunohistochemistry were used to measure cytokine responses. FINDINGS After cold challenge, untreated patients with FCAS developed rash, fever, and arthralgias within 1-4 h. Significant increases in serum concentrations of interleukin 6 and white-blood-cell counts were seen 4-8 h after cold challenge. Serum concentrations of interleukin 1 and cytokine mRNA in peripheral-blood leucocytes were not raised, but amounts of interleukin 1 protein and mRNA were high in affected skin. IL-1Ra administered before cold challenge blocked symptoms and increases in white-blood-cell counts and serum interleukin 6. INTERPRETATION The ability of IL-1Ra to prevent the clinical features and haematological and biochemical changes in patients with FCAS indicates a central role for interleukin 1beta in this disorder. Involvement of cryopyrin in activation of caspase 1 and NF-kappaB signalling suggests that it might have a role in many chronic inflammatory diseases. RELEVANCE TO PRACTICE These findings support a new therapy for a disorder with no previously known acceptable treatment. They also offer insights into the role of interleukin 1beta in more common inflammatory diseases.

Inhibition of airway remodeling in IL-5–deficient mice

To determine the role of IL-5 in airway remodeling, IL-5-deficient and WT mice were sensitized to OVA and challenged by repetitive administration of OVA for 3 months. IL-5-deficient mice had significantly less peribronchial fibrosis (total lung collagen content, peribronchial collagens III and V) and significantly less peribronchial smooth muscle (thickness of peribronchial smooth muscle layer, alpha-smooth muscle actin immunostaining) compared with WT mice challenged with OVA. WT mice had a significant increase in the number of peribronchial cells staining positive for major basic protein and TGF-beta. In contrast, IL-5-deficient mice had a significant reduction in the number of peribronchial cells staining positive for major basic protein, which was paralleled by a similar reduction in the number of cells staining positive for TGF-beta, suggesting that eosinophils are a significant source of TGF-beta in the remodeled airway. OVA challenge induced significantly higher levels of airway epithelial alphaVbeta6 integrin expression, as well as significantly higher levels of bioactive lung TGF-beta in WT compared with IL-5-deficient mice. Increased airway epithelial expression of alphaVbeta6 integrin may contribute to the increased activation of latent TGF-beta. These results suggest an important role for IL-5, eosinophils, alphaVbeta6, and TGF-beta in airway remodeling.

Accumulation of Peribronchial Mast Cells in a Mouse Model of Ovalbumin Allergen Induced Chronic Airway Inflammation: Modulation by Immunostimulatory DNA Sequences

Few peribronchial mast cells are noted either in the lungs of naive mice or in the lungs of OVA-sensitized mice challenged acutely with OVA by inhalation. In this study, we demonstrate that OVA-sensitized mice exposed to repetitive OVA inhalation for 1–6 mo have a significant accumulation of peribronchial mast cells. This accumulation of peribronchial mast cells is associated with increased expression of the Th2 cell-derived mast cell growth factors, including IL-4 and IL-9, but not with the non-Th2 cell-derived mast cell growth factor, stem cell factor. Pretreating mice with immunostimulatory sequences (ISS) of DNA containing a CpG motif significantly inhibited the accumulation of peribronchial mast cells and the expression of IL-4 and IL-9. To determine whether mast cells express Toll-like receptor-9 (TLR-9; the receptor for ISS), TLR-9 expression by mouse bone marrow-derived mast cells (MBMMCs) was assessed by RT-PCR. MBMMCs strongly expressed TLR-9 and bound rhodamine-labeled ISS. However, incubation of MBMMCs with ISS in vitro neither inhibited MBMMC proliferation nor inhibited Ag/IgE-mediated MBMMC degranulation, but they did induce IL-6. Overall these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express increased levels of mast cell growth factors in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s), which include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.

Systemic Administration of Immunostimulatory DNA Sequences Mediates Reversible Inhibition of Th2 Responses in a Mouse Model of Asthma

This study investigated whether immunostimulatory DNA sequences (ISS) induce a transient or sustained inhibition of Th2 responses to inhaled antigen. We sensitized mice with subcutaneous injections to develop a Th2 response to ovalbumin (ova) and then administered a dose of ISS prior to ova inhalation challenge. Mice were then rechallenged with ova by inhalation a second time at varying time points after the first ova inhalation (1 to 8 weeks later) to determine whether the ISS dose administered prior to the first ova inhalation protected against a subsequent second ova inhalation challenge. A single dose of ISS inhibited the Th2 response to the first inhalation of ova antigen, as well as 4 weeks later to the second inhalation of ova. However, ISS did not inhibit a Th2 response to the second inhalation of ova 8 weeks later. The reversible inhibition of Th2 responses at 8 weeks suggests the need for repeated ISS administration at monthly intervals.

Immunostimulatory DNA Reverses Established Allergen-Induced Airway Remodeling

To determine whether immunostimulatory sequences of DNA (ISS) can reverse established airway remodeling, mice that had developed airway remodeling following 3 mo of repetitive OVA challenges, were treated with ISS for 1–3 mo. Systemic administration of ISS to mice that had already developed established airway remodeling significantly reduced the degree of airway collagen deposition (assessed by lung collagen content, peribronchial trichrome staining, and immunostaining with anticollagen type III and type V Abs). ISS reduced bronchoalveolar lavage and lung levels of TGF-β1 and reduced the number of TGF-β1-positive eosinophils and TGF-β1-positive mononuclear cells recruited to the airway. In vitro studies demonstrated that ISS inhibited TGF-β1 expression by macrophages (RAW 264.7 cell line and bone marrow-derived macrophages). In addition, ISS significantly reduces lung levels of expression of the chemokine thymus- and activation-regulated chemokine, as well as the number of peribronchial CD4+ lymphocytes that express Th2 cytokines that promote peribronchial fibrosis. Overall, these studies demonstrate that ISS can reverse features of airway collagen deposition by reducing levels of lung TGF-β1, as well as by reducing levels of the chemokine thymus- and activation-regulated chemokine and the numbers of peribronchial CD4+ lymphocytes that drive the ongoing Th2 immune response.

Eosinophil tissue recruitment to sites of allergic inflammation in the lung is platelet endothelial cell adhesion molecule independent.

Platelet endothelial cell adhesion molecule (PECAM or CD31) is a cell adhesion molecule expressed on circulating leukocytes and endothelial cells that plays an important role in mediating neutrophil and monocyte transendothelial migration in vivo. In this study, we investigated whether eosinophils, like neutrophils and monocytes, utilize PECAM for tissue recruitment to sites of allergic inflammation in vivo. Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis. RT-PCR studies demonstrate that eosinophils like neutrophils express the six extracellular domains of PECAM. Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM. The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs did not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, and airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo. Thus, in contrast to studies that have demonstrated that neutrophil and monocyte tissue recruitment is PECAM dependent, these studies demonstrate that eosinophil tissue recruitment in vivo in this model is PECAM independent.

Plasmid DNA encoding the respiratory syncytial virus G protein protects against RSV-induced airway hyperresponsiveness

Respiratory syncytial virus (RSV) is an important cause of childhood respiratory disease as well as exacerbations of asthma. Although previous studies have demonstrated that a DNA vaccine encoding the RSV G protein can inhibit RSV replication in mouse models of RSV infection, studies have not been performed to determine whether a DNA vaccine encoding the RSV G protein can protect against RSV induced mucus expression and airway hyperresponsiveness which was the focus of this study. The DNA-G vaccine we constructed significantly inhibited RSV viral replication, mucus expression, and importantly was associated with inhibition of RSV induced airway responsiveness.