Puneeta Nath

IL-4 and IL-13 Form a Negative Feedback Circuit with Surfactant Protein-D in the Allergic Airway Response

The innate immune molecule surfactant protein-D (SP-D) plays an important regulatory role in the allergic airway response. In this study, we demonstrate that mice sensitized and challenged with either Aspergillus fumigatus (Af) or OVA have increased SP-D levels in their lung. SP-D mRNA and protein levels in the lung also increased in response to either rIL-4 or rIL-13 treatment. Type II alveolar epithelial cell expression of IL-4Rs in mice sensitized and challenged with Af, and in vitro induction of SP-D mRNA and protein by IL-4 and IL-13, but not IFN-γ, suggested a direct role of IL-4R-mediated events. The regulatory function of IL-4 and IL-13 was further supported in STAT-6-deficient mice as well as in IL-4/IL-13 double knockout mice that failed to increase SP-D production upon allergen challenge. Interestingly, addition of rSP-D significantly inhibited Af-driven Th2 cell activation in vitro whereas mice lacking SP-D had increased numbers of CD4+ cells with elevated IL-13 and thymus- and activation-regulated chemokine levels in the lung and showed exaggerated production of IgE and IgG1 following allergic sensitization. We propose that allergen exposure induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which in turn, prevents further activation of sensitized T cells. This negative feedback regulatory circuit could be essential in protecting the airways from inflammatory damage after allergen inhalation.

Allergen-induced inflammation and airway epithelial and smooth muscle cell proliferation: role of Jun N-terminal kinase.

Chronic cellular inflammation and airway wall remodelling with subepithelial fibrosis and airway smooth muscle (ASM) cell hyperplasia are features of chronic asthma. Jun N‐terminal kinase (JNK) may be implicated in these processes by regulating the transcriptional activity of activator protein (AP)‐1. We examined the effects of an inhibitor of JNK, SP600125 (anthra [1,9‐cd] pyrazole‐6 (2 H)‐one), in a model of chronic allergic inflammation in the rat. Rats sensitised to ovalbumin (OA) were exposed to OA‐aerosol every third day on six occasions and were treated with SP600125 (30 mg kg−1 b.i.d; 360 mg in total) for 12 days, starting after the second through to the sixth OA exposure. We measured eosinophilic and T‐cell inflammation in the airways, proliferation of ASM cells and epithelial cells by incorporation of bromodeoxyuridine (BrdU), and bronchial responsiveness to acetylcholine. SP600125 significantly reduced the number of eosinophils (P<0.05) and lymphocytes (P<0.05) in bronchoalveolar lavage fluid, suppressed eosinophilic (P<0.05) and CD2+ T‐cell (P<0.05) infiltration within the bronchial submucosa, and the increased DNA incorporation in ASM (P<0.05) and epithelial cell incorporation (P<0.05). SP600125 did not alter bronchial hyper‐responsiveness observed after chronic allergen exposure. Pathways regulated by JNK positively regulate ASM cell proliferation and allergic cellular inflammation following chronic allergen exposure.

Attenuation of Ozone-Induced Airway Inflammation and Hyper-Responsiveness by c-Jun NH2 Terminal Kinase Inhibitor SP600125

Ozone has potent oxidizing properties, and exposure to ozone causes airway hyper-responsiveness (AHR) and lung inflammation. We determined the importance of c-Jun NH2 terminal kinase (JNK), a member of the mitogen-activated protein kinase pathway, in ozone-induced AHR and inflammation. SP600125 [anthra[1,9-cd] pyrazol-6 (2H)-one], a specific JNK inhibitor (30 mg/kg) or vehicle, was administered by intraperitoneal injection before and after ozone exposure (3 ppm for 3 h). SP600125 significantly reduced total cells, and neutrophils in bronchoalveolar fluid recovered at 20 to 24 h after exposure and inhibited ozone-induced AHR. Ozone exposure induced activation of JNK in the lung as measured by the expression of phosphorylated-c-Jun, an effect abolished by SP600125. Gene-microarray analysis revealed that ozone increased the expression of over 400 genes by more than 2-fold, including interleukin-6 (IL-6), CXCL1 (keratinocyte cytokine), and CCL2 (monocyte chemoattractant protein-1). SP600125 modulated the expression of a subset of 29 ozone-induced genes; IL-6 and CCL2 expression were further increased, whereas the expression of metallothionein 1, hemopexin, and mitogen-activated 3 kinase 6 was decreased in SP600125-treated ozone-exposed mice. Changes in mRNA for IL-6, CXCL1, and CCL2 were confirmed by real-time polymerase chain reaction. Ozone also decreased the expression of over 500 genes, with the most potent effect on angiopoietin-1. SP600125 modulated the expression of 15 of these genes, and in particular, SP600125 reversed ozone-induced decrease in expression of the redox-sensitive transcription factor, hypoxia-induced factor-1α. This study highlights an important role for JNK in response to oxidative stress through modulation of specific inflammatory and redox mediators. Inhibition of JNK with small molecule kinase inhibitors may be a means of reducing ozone-induced inflammation and AHR.

Complete inhibition of allergic airway inflammation and remodelling in quadruple IL‐4/5/9/13−/− mice

Background T‐helper type 2 (Th2)‐derived cytokines such as IL‐4, IL‐5, IL‐9 and IL‐13 play an important role in the synthesis of IgE and in the promotion of allergic eosinophilic inflammation and airway wall remodelling.

Effect of an inhibitor of Jun N-terminal protein kinase, SP600125, in single allergen challenge in sensitized rats

Jun N‐terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9‐cd] pyrazol‐6 (2H)‐one), a novel inhibitor of JNK in a model of asthma. Brown‐Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen‐induced, increased activity of phosphorylated c‐jun but not of phosphorylated‐MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0·04), lymphocyte (P < 0·05), eosinophil (P < 0·04) and neutrophil (P < 0·005) numbers in bronchoalveolar lavage. Eosinophil and T‐cell accumulation in the airways, mRNA expression for interleukin‐1β, tumour necrosis factor‐β, interleukin‐3, interleukin‐4 and interleukin‐5, serum levels of allergen‐specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen‐activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.

Role of cathepsin S in ozone-induced airway hyperresponsiveness and inflammation

Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20-24 h following exposure, AHR was measured at 20-24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg(-1) p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (-logPC100 vehicle: -0.70+/-0.12, n=8 vs. cathepsin S inhibitor: -1.30+/-0.06, P<0.001, n=8) at 20-24 h and BAL neutrophilia at 3 h and 20-24 h (P<0.05, n=6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-alpha and IFN-gamma. Compound A reduced IL-6 at 3 h and 20-24 h (P<0.05, n=5) and TNF-alpha, at 20-24 h (P<0.05, n=6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.

Inhibition of citric acid- and capsaicin-induced cough by novel TRPV-1 antagonist, V112220, in guinea-pig

BackgroundCough reflex can be induced by the pepper extract capsaicin and by low pH in guinea-pig airways. Transient receptor potential vanniloid-1 (TPRV-1) is expressed in the sensory and afferent nerve fibres in airways.ObjectiveWe hypothesized that a novel pyridazinylpiperazine analog TPRV-1 inhibitor can effectively reduce cough reflex stimulated by citric acid and capsaicin.MethodsGuinea pigs were injected with specific TPRV-1 inhibitor, V112220, a pyridazinylpiperazine analog of N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide (BCTC) (3 mg/kg) intra-peritoneally. One hour before cough response assessment. Coughs were recorded using a recorder system that identified cough sound and accompanying expiratory flows, distinct from sneezes. Guinea-pigs exposed to citric acid (0.4 M) and to capsaicin (10-4M) aerosols, in succession separately by 2 hours.ResultsV112220 significantly inhibited the number of coughs induced by citric acid (73 ± 11%, p < 0.01) and capsaicin (70 ± 9.4%, p < 0.05) compared to vehicle control.ConclusionA novel pyridazinylpiperazine analog TPRV-1 inhibitor can inhibit the cough reflex, induced by both low pH and capsaicin, suggesting that it could be clinically beneficial in treatment of cough.

Modulation of ozone-induced airway hyperresponsiveness and inflammation by interleukin-13

The present study aimed to determine whether the T-helper cell type 2-derived cytokines, interleukin (IL)-4 and -13, can modulate the lung response to ozone exposure. IL-13-/-, IL-4/13-/- and IL-13-overexpressing transgenic (Tg) mice were exposed to ozone (3 ppm; 3 h) or air. Wild-type (Wt) Balb/c mice and transgenic-negative littermates (IL-13Wt) were used as controls for gene-deficient and IL-13Tg mice, respectively. IL-4/13-/- and IL-13-/- mice developed a lesser degree of ozone-induced airway hyperresponsiveness (AHR) while IL-13Tg mice developed a greater degree of AHR compared with ozone-exposed wild-type or IL-13Wt mice, respectively. Ozone caused a time-dependent increase of bronchoalveolar lavage (BAL) neutrophils and macrophages in wild-type mice, maximal at 20–24 h, which was attenuated in the IL-13-/- and IL-4/13-/- mice. In IL-13Tg mice, there was a greater increase in BAL neutrophils after ozone exposure compared with IL-13Wt mice. Using quantitative real-time PCR, ozone-induced mRNA expression for IL-6 and keratinocyte chemokine was further enhanced in IL-13-/- and IL-4/13-/- mice, and was inhibited in IL-13Tg mice. Macrophage inflammatory protein (MIP)-3α/CCL20 expression was enhanced after ozone exposure in wild-type mice, inhibited in IL-13-/- and IL-4/13-/- mice, while in IL-13Tg mice it was enhanced. A similar pattern of expression was observed with lipopolysaccharide-induced cytokine (LIX/CXCL5/ENA-78) expression. In conclusion, interleukin-13 augments ozone-induced airway hyperresponsiveness and neutrophilic inflammation, possibly through modulation of certain cytokines induced by ozone exposure.

Resolution of allergic airways inflammation but persistence of airway smooth muscle proliferation after repeated allergen exposures

Background Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall.