Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

SummaryBackgroundCCR3 is the cognate receptor for major human eosinophil chemoattractants from the eotaxin family of proteins that are elevated in asthma and correlate with disease severity.ObjectiveThis proof‐of‐mechanism study examined the effect of AXP1275, an oral, small‐molecule inhibitor of CCR3, on airway responses to inhaled allergen challenge.MethodsTwenty‐one subjects with mild atopic asthma and documented early and late asthmatic responses to an inhaled aeroallergen completed a randomized double‐blind cross‐over study to compare early and late allergen‐induced asthmatic responses, methacholine PC20, blood and sputum eosinophils and exhaled nitric oxide after 2 weeks of treatment with once‐daily doses of AXP1275 (50 mg) or placebo.ResultsThere was a significant increase in methacholine PC20 after 12 days of AXP1275 treatment compared to placebo (increase of 0.92 doubling doses versus 0.17 doubling doses, P = .01), but this protection was lost post‐allergen challenge. There was no effect of AXP1275 on allergen‐induced late asthmatic responses, or eosinophils in blood and sputum. The early asthmatic response and exhaled nitric oxide levels were slightly lower with AXP1275, but this did not reach statistical significance. The number of subjects who experienced treatment‐emergent adverse events while receiving AXP1275 was comparable placebo.Conclusions & Clinical RelevanceAXP1275 50 mg administered daily was safe and well tolerated, and there was no difference in the type, severity or frequency of treatment‐emergent adverse events in subjects while receiving AXP1275 compared to placebo. AXP1275 increased the methacholine PC20; however, the low and variable exposure to APX1275 over a short treatment period may have contributed to poor efficacy on other outcomes.

We have identified a novel serine/threonine protein kinase, MAP3K19, whose expression in normal lung was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. We have also found MAP3K19 to be expressed in multiple, primary NSCLC tumor samples, as well as human lung cancer cell lines, including A549. The kinase is transcriptionally upregulated in cells upon various types of cell stress, including oxidative, endoplasmic reticulum and osmotic stress. Using two different murine xenograft models, we assayed the role of MAP3K19 to inhibit the growth of either primary human NSCLC tumors and A549 cells using small molecule inhibitors. The ability of i.v. injected A549 cells to colonize and grow in the lung was significantly reduced in mice that received orally administered, selective MAP3K19 inhibitors. Similar results were observed in a subcutaneous xenograft model, as A549 tumor cell growth was inhibited by both MAP3K19 antagonists and other standard of care kinase inhibitors. These studies also showed an additive anti-proliferative effect when gefitinib or sorafenib and MAP3K19 inhibitors were co-administered. Importantly, xenograft models using primary human NSCLC tumors implanted subcutaneously in immunodeficient mice showed a statistically significant inhibition of tumor growth when the mice were treated with the orally administered MAP3K19 antagonists. IHC analysis of the tumors showed that mice treated with the MAP3K19 inhibitors also had decreased levels of Ki-67, c-myc, p27 and phospho-Bim staining and increased caspase-3 staining. These results suggest a molecular mechanism by which MAP3K19 may inhibit tumor cell growth, and further suggest that inhibition of MAP3K19 either by itself or in combination with other therapies may represent a new avenue for the treatment of NSCLC. The clinical development of the MAP3K19 inhibitor is expected to initiate Phase I clinical trials in early 2017.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients.

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

MAP3K19 (RC Kinase) is a novel nuclear kinase that is expressed in healthy lung by macrophages and type II epithelial cells, upregulated in IPF lungs, and commonly found in atypical epithelium adjacent to fibroblastic foci. Consistent with this cellular distribution, late-stage pre-clinical small molecule inhibitors of MAP3K19 caused significant reductions in inflammatory infiltrate, extra-cellular matrix deposition and hydroxyproline production in a murine xenograft model of IPF. In the murine bleomycin model, prophylactic or therapeutic oral administration of a MAP3K19 antagonist compound significantly decreased inflammation, fibrosis and collagen content. In contrast, Pirfenidone only significantly affected lung collagen levels. To understand the mechanism of action of specific MAP3K19 inhibitors, we examined whether the TGF-β signaling pathway was impacted. An examination of IPF patient-derived myofibroblast cell lines, primary human macrophages, and numerous MAP3K19+ human cell lines showed that inhibition of MAP3K19 resulted in a significant decrease in the nuclear accumulation of P-Smad-2 and P-Smad-3 upon TGF-β stimulation. Pirfenidone had minimal effects. Inhibition of MAP3K19 did not affect Smad-4 levels, and cytoplasmic levels of Smad-2 and Smad-3 were also unaffected. Inhibition of MAP3K19 also blocked TGF-β-induced gene transcription of various EMT molecules. Surprisingly, inhibition of MAP3K19 also inhibited TGF-β-induced nuclear translocation of Notch. These results suggest that MAP3K19 may act as a regulator of nuclear translocation of various signaling molecules involved in transcription, and provides a mechanism to explain how MAP3K19 inhibition may provide therapeutic benefit in IPF.

We have developed a novel therapeutic inhibitor against the chemokine receptor CCR3 for allergic asthma that significantly inhibited the MCh PC20 response in a Phase IIa trial in mild-to-moderate asthmatics. In vivo, AXP1275 had shown significant efficacy in reducing inflammatory mediators and cellular infiltrates in non-human primate models of allergic asthma, and, surprisingly, had also inhibited airways hyperreactivity and methacholine constriction responses (PC150). In addition, inhibition was seen with ex-vivo carbachol (CCh)-induced constriction in human lung tissue. In order to discern pharmacologically-relevant mechanisms for inhibition of CCh-induced lung constriction by AXP1275, we have investigated the receptor pharmacology of CCR3 in human lung tissue. Immunohistochemically, CCR3 is present on epithelium and smooth muscle, and is co-localized with the muscarinic M3 receptor. In receptor transfectants, CCL11 and CCh cross-sensitize agonist-induced calcium mobilization, suggesting cooperativity between CCR3 and M3. AXP1275 and muscarinic receptor antagonists inhibit responses in a similarly-cooperative manner. Bioluminescence resonance energy transfer (BRET) assays demonstrated that CCR3 and M3 heterodimerize, and this was further supported by receptor co-immunoprecipitation assays. Receptor heterodimerization of CCR3 and M3 on non-leukocytic cells in vivo may provide a plausible explanation for the efficacy of CCR3-selective inhibitors on the hyperreactivity response in asthma.

Proteomics is a powerful tool to ascertain which proteins are differentially expressed in the context of disease. We have used this approach on inflammatory cells obtained from patients with asthma to ascertain whether novel drugs targets could be illuminated and to investigate the role of any such target in a range of in vitro and in vivo models of inflammation.A proteomic study was undertaken using peripheral blood mononuclear cells from mild asthmatic subjects compared with healthy subjects. The analysis revealed an increased expression of the intracellular kinase, mitogen activated protein kinase (MKK3), and the function of this protein was investigated further in preclinical models of inflammation using MKK3 knockout mice.We describe a 3.65 fold increase in the expression of MKK3 in CD8(+) T lymphocytes obtained from subjects with asthma compared with healthy subjects using a proteomic approach which we have confirmed in CD8(+), but not in CD4(+) T lymphocytes or human bronchial epithelial cells from asthmatic patients using a Western blot technique. In wild type mice, bacterial lipopolysaccharide (LPS) caused a significant increase in MKK3 expression and significantly reduced airway neutrophilia in MKK3(-/-) mice (median, 25, 75% percentile; wild/LPS; 5.3 (0.7-9.9) x 10(5) cells/mL vs MKK3(-/-)/LPS; 0 (0-1.9) x 10(5) cells/mL, P < 0.05). In contrast, eosinophilia in sensitized wild type mice challenged with allergen (0.5 (0.16-0.65) x 10(5) cells/mL) was significantly increased in MKK3(-/-) mice (2.2 (0.9-3.5) x 10(5) cells/mL, P < 0.05).Our results suggest that asthma is associated with MKK3 over-expression in CD8(+) cells. We have also demonstrated that MKK3 may be critical for airway neutrophilia, but not eosinophilia, suggesting that this may be a target worthy of further consideration in the context of diseases associated with neutrophil activation such as severe asthma and COPD. (C) 2014 Elsevier Ltd. All rights reserved.

RC kinase is a novel serine/threonine kinase, expressed predominantly by pulmonary macrophage and type II bronchial epithelial cells. Examination of BAL macrophage mRNA isolated from COPD and IPF patients showed increased expression of RC kinase compared to normal smokers and non-smoking patients. In vitro studies showed an upregulation of RC kinase upon cell stress, including oxidative stress. This correlated with an increase of NF-kb activity and IL-8 production that could be inhibited by RC kinase siRNA or small molecule antagonists. Biochemical analysis revealed that RC kinase is predominantly a nuclear protein and interacts with p27, a CDK inhibitor protein, and the NF-kb pathway. We have developed specific, extremely potent and orally administered small molecule antagonists of RC kinase with a favorable ADME profile. Mice treated with these compounds in an acute model of COPD showed significant reductions in the number of BAL neutrophils and the chemokine KC. Similar results were seen in mice treated with RC kinase siRNA. In a chronic (5 month) cigarette smoke exposure model, the small molecule antagonists were effective in both reducing inflammation and significantly limiting alveoli destruction. Inhibition of RC kinase in an acute cigarette smoke and viral exacerbation model reduced BAL neutrophils, inhibited cytokines such as IL-6 and significantly reduced viral titers. In a murine xenograft model of IPF, inhibition of RC kinase by small molecules or siRNA caused significant reductions in the inflammatory infiltrate, hydroxyproline production and collagen deposition in the lungs. Additionally, mRNA levels of collagens 1 & 3, smooth muscle actin and fibronectin were also reduced.

printing supported by . Visit Chiesi at Stand B2.10 MONDAY, SEPTEMBER 3RD 2012 correlated with the production of IL-8. In acute (4 day) and sub-chronic (14 day) cigarette smoke-induced murine models of COPD, treatment with either intratracheally delivered RC kinase siRNA or orally administered novel and specific small molecule inhibitors caused a significant reduction in BAL neutrophilia, as well as decreased levels of KC and CCL-20. There was also a marked reduction in the amount of pulmonary inflammation. In a murine adoptive transfer model of idiopathic pulmonary fibrosis, both siRNA and small molecule antagonist treatment significantly inhibited hydroxyproline production, inflammation and cellular and biochemical markers of fibrosis. Taken together, these results strongly suggest that inhibition of RC kinase may provide a novel therapeutic approach for the treatment of COPD and IPF. P2125 Statins worse pulmonary fibrosis through enhancing NLRP3 inflammasome activation Jin-Fu Xu1,2, George R. Washko2, Hui-Ping Li1, Augustine M.K. Choi2, Gary M. Hunninghake2. 1Department of Pulmonary Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China; 2Pulmonary and Critical Care Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States The role of statins is controversial. To evaluate the association between statin use and ILD. We used regression analyses to evaluate the association between statin use and interstitial lung abnormalities (ILA) in a large cohort of smokers from COPDGene. Next, we evaluated the effect of statin pretreatment on bleomycin-induced fibrosis in mice and explored the mechanism behind these observations in vitro. In COPDGene, 38% of subjects with ILA were taking statins compared to 27% of subjects without ILA. Statin use was positively associated in ILA (odds ratio [OR] 1.60, 95% confidence interval [CI] 1.03-2.50, P=0.04) after adjustment for covariates including a history of high cholesterol or coronary artery disease. This association was modified by the hydrophilicity of statin and the age of the subject. Next, we demonstrate that statin administration aggravates lung injury and fibrosis in bleomycin-treated mice. Statin pretreatment enhances caspase-1-mediated immune responses in vivo and in vitro; the latter responses were abolished in bone marrow-derived macrophages (BMDM) isolated from Nlrp3-/and Casp1-/mice. Finally, we provide further insights by demonstrating that statins enhance NLRP3-inflammasome activation by increasing mitochondrial reactive oxygen species generation in macrophages. Statin use is associated with ILA among smokers in the COPDGene study and enhances bleomycin-induced lung inflammation and fibrosis in the mouse through a mechanism involving enhanced NLRP3-inflammasome activation. Our findings suggest that clinicians should be aware that radiological evidence of ILD can develop in some COPD patients treated with statins. P2126 Effects of combination of PI3Kγ and δ inhibitors on airway hyperresponsiveness in tobacco smoke-exposed mice Yasuo Kizawa1, Genki Kimura1, Keitaro Ueda1, Yuji Watanabe1, Shouichi Eto1, Tadashi Kusama1, Kazuhiro Ito2. 1Physiology and Anatomy, Nihon University School of Pharmacy, Funabashi, Chiba, Japan; 2Airway Disease Section, NHLI, Imperial College, London, United Kingdom PI3K δ and γ are known to be involved in inflammatory cell functions. We recently found upregulation of PI3Kδ in lung tissue of COPD patients and ability of a PI3Kδ inhibitor on restoration of steroid sensitivity in airway inflammation in tobacco-smoke (TS) exposed mice. Superior effects of combination of PI3Kγ and δ inhibitors to each inhibitor alone on airway inflammation in TS-exposed mice were also observed. The aim of this study is to evaluate role of PI3Kγ and PI3Kδ on airway hyperresponsiveness (AHR) in TS-exposed mice. A/J mice were exposed to TS for 11 days and IC87114 (IC), AS604850 (AS) and/or fluticasone propionate (FP) were administered intranasally twice a day for 3 days after the last TS exposure. Airway responsiveness was determined as the increment of airway resistance ( [sRaw/TV]) before and 1 min after histamine inhalation at 24 h after the last drug dosing. The effects of the PI3K inhibitors on the contractile response to carbachol in guinea-pig tracheal smooth muscle preparation were also evaluated by the isometric tension recording. The concentration-response curve of carbachol was shifted to rightward and reduced the maximal response by AS (10-100 μM), in contrast, the effects of IC (100 μM) was limited in the tracheal smooth muscle. The AHR induced by TS was significantly reduced by AS (4 mg/ml; by 56% inhibition) and IC (4 mg/ml; 43%) alone. The inhibitory effects were enhanced by combination treatment of AS and IC (69%). Moreover, the combination of IC and FP showed stronger inhibition (96%) on the AHR.Considering with our previous findings, the combination of a PI3Kδ or PI3Kγ/δ inhibitor with corticosteroid may offer potential treatment of COPD. P2127 A robust translational model of acute exacerbations in the tobacco-smoke and poly IC treated mouse Vincent Russell, Paul Woodman, Andrew Connolly, Dianne Spicer, Joanna Dlugozima, Alan Young. Pharmacology, Argenta, Stoke Court, Slough, United Kingdom Exposure to tobacco smoke (TS) for 4 days induces steroid-insensitive lung inflammation in mice. The effect of adding the viral mimetic poly IC (PIC) to TS-exposed mice was examined. Methods: Mice were exposed daily to either TS or air for 4d. Saline or PIC was administered intra-nasally. The time course of lung inflammation was examined 4-120hrs after the last exposure and cell numbers measured in the BAL fluid. The acute effects of oral Dexamethasone (DEX 0.3mg/kg) or Roflumilast (ROF 5mg/kg) on the peak inflammation were examined. The effects of DEX on the kinetics of the enhanced inflammation were also examined. Results: TS caused a lung inflammation which was inhibited by ROF but not by DEX. PIC alone induced an inflammation that was not inhibited by DEX or ROF. Dosing PIC in addition to TS induced an exaggerated response that was significantly greater than the additive effect of the two stimuli. The enhanced response peaked 24hrs after the last exposure then slowly declined. Neutrophils were predominant over the first 48 hrs. Macrophage numbers increased at 24-72hrs and lymphocyte numbers peaked at 48-72hrs. The peak inflammation after TS/PIC exposure was significantly inhibited by ROF (53%, p<0.05) and DEX (56%, p<0.05), in contrast to the lack of efficacy of DEX against TS or PIC alone. A single dose of DEX after the last exposure reduced the exaggerated response over the entire 120hr study period, but did not fully resolve the inflammation. Conclusions: TS exposure for 4 days induced a steroid-insensitive lung inflammation. Addition of PIC markedly enhanced the inflammatory response which was sensitive to both steroids and roflumilast, mimicking features of human COPD. P2128 Inhaled cationic salts modulate macrophage function to reduce inflammation during LPS induced lung injury S.P. Arold, E.L. Berry, D. Rosa, F.A. Saia, S. Kong, P.L. Wright, R.W. Clarke, D.L. Hava. Research, Pulmatrix, Lexington, MA, United States Pulmatrix is developing PUR118 as a host-targeted, dry powder therapy based on the inhalation of calcium salts for acute exacerbation (AE) control in chronic obstructive pulmonary disease and other inflammatory lung disease. Preclinical data suggest that this approach is effective against an array of pathogens and also reduces inflammation resulting from environmental stimuli such as tobacco smoke. We hypothesized that this treatment could be efficacious in reducing lipopolysaccharide (LPS) induced lung inflammation by modulating the function of pulmonary macrophages. Mice were exposed to nebulized LPS (Pseudomonas aeruginosa) and PUR118, was delivered via whole body exposure 1h post-LPS challenge. Four hours after LPS exposure inflammatory cell counts and chemokine and cytokine concentrations were determined in BAL. PUR118 treatment decreased total inflammatory cell counts and neutrophil counts in the BAL fluid of LPS challenged mice and correlated with reduced KC, IL-6 and TNF-α in BAL fluid. Separately, peritoneal macrophages were isolated from naïve mice and challenged with LPS in media supplemented with calcium to simulate conditions thought to be found in lung fluid lining after PUR118 treatment. Inflammatory mediator secretion and gene expression were determined 2h post LPS exposure. Macrophages stimulated with LPS in the presence of calcium exhibited a dose dependent decrease in KC, IL-6 and TNF-α secretion as well as reduced gene expression for these inflammatory mediators. These data suggest that PUR118 can act through macrophages to reduce lung inflammation and may reduce the risk of AE caused by infections during chronic lung disease. P2129 Inhaled calcium salts reduce expression of inflammatory mediators associated with tobacco smoke exposure to reduce airway inflammation P.L. Wright1, P. Woodman2, D. Spicer2, J. Kenyon1, P. Okerholm1, V. Russell2, R.W. Clarke1, D.L. Hava1. 1Research, Pulmatrix, Lexington, MA, United States; 2Respiratory, Argenta, Slough, United Kingdom PUR118, an inhaled calcium based dry powder (DP) formulation exhibits preclinical anti-inflammatory and anti-infective activity. PUR118 may provide a novel approach for acute exacerbation control in patients with COPD and CF where the combination of underlying inflammation and pathogen infection result in reduced lung function and quality of life. The goal of this study was to evaluate the impact of PUR118 on gene expression in lung samples from a tobacco smoke (TS) exposure model. Mice were exposed to TS for 4d and treated with PUR118 or DP control 1h prior to TS. Mice were euthanized 4h after the last TS exposure and BAL and lung RNA were collected for cell counts, protein levels and QPC

Aim: CCR3 has historically been associated with the functional responses of eosinophils in various models of allergic disease. The goal of this study was to determine whether pre-treatment with CCR3 inhibitors via oral and inhaled routes attenuates asthma responses in ascaris sensitized Cynomolgus monkeys. Methods: Animals received a CCR3 antagonist given orally (AP0), or by inhalation (AR1) once or twice a day for 10 or up to 21 days prior to inhaled ascaris challenge. Changes in airway function (immediate and methacholine [MCh]) and inflammation (BAL & blood cells) were evaluated. Results: Oral (AP0 5 mg/kg; BID 10 days) or inhaled (AR1 860 μg; BID 7 days) treatment showed a trend towards a reduced immediate ascaris and MCh response but did not reach statistical significance. A longer oral treatment (AP0 3 mg/kg; QD 20 days) resulted in a significant reduction in both immediate bronchoconstriction and AHR. This was not associated with a consistent effect on BAL or blood eosinophils, but reduced the lymphocyte, macrophage and mast cell numbers. AP0 given in combination with inhaled fluticasone (79 μg BID) did not yield in a significant additive or synergistic effect on airway function but did lead to a greater reduction in BAL and blood eosinophils than AP0 or fluticasone alone. Conclusion: Treatment with a CCR3 inhibitor in the non-human primates, Ascaris model of asthma, shows that a number of critical parameters can be affected which are significantly different to alterations in the recruitment of eosinophils. Overall, these observations suggest that CCR3 inhibition may have more globally-beneficial responses in an asthmatic setting than previously appreciated.

We have characterized a novel serine/threonine protein kinase, called RC kinase, whose expression is upregulated in COPD patients. Examination of RC kinase mRNA tissue distribution showed a limited expression pattern restricted mainly to the lungs and trachea. Immunohistochemical analysis with a monoclonal antibody revealed expression in CD68+ alveolar macrophage and bronchial epithelial cells. Various cell lines upregulated RC kinase expression upon exposure to cigarette smoke extract, or conditions of oxidative or endoplasmic reticulum stress, and this correlated with the production of IL-8. In acute (4 day) and sub-chronic (14 day) cigarette smoke-induced murine models of COPD, treatment with either intra-tracheally delivered RC kinase siRNA or orally administered novel and specific small molecule inhibitors caused a significant reduction in BAL neutrophilia, as well as decreased levels of KC and CCL-20. There was also a marked reduction in the amount of pulmonary inflammation. In a murine adoptive transfer model of idiopathic pulmonary fibrosis, both siRNA and small molecule antagonist treatment significantly inhibited hydroxyproline production, inflammation and cellular and biochemical markers of fibrosis. Taken together, these results strongly suggest that inhibition of RC kinase may provide a novel therapeutic approach for the treatment of COPD and IPF.

MKK3 is a member of the p38 MAPK signalling pathway. Studies have shown that MKK3 is an important factor in non allergic inflammatory and Th1 responses. Less is known about the role of MKK3 in allergic inflammation and allergic diseases. Proteomic analysis was performed on peripheral blood cells obtained from 22 healthy and 18 allergic asthmatic patients. Lysates from purified lymphocytes were separated by SDS-PAGE and the protein inventories of each sample were identified using mass spectrometry. A 3.65 fold increase in the expression of MKK3 was observed in CD8 + T lymphocytes from asthmatic in relation to healthy volunteers, (% vol protein abundance). Western blot (WB) analysis showed MKK3 expression in human CD4 + lymphocytes, human endothelial cells (HUVECs) and human epithelial cells (HBECs) but not in neutrophils or eosinophils. However, using densitometric analysis we found no differences between healthy and asthmatic subjects (n=10) in any of the cell types analysed. Real time PCR studies showed no expression of MKK3 isoform C in any tissue or cell type. However, large quantities of isoform B gene expression were found in neutrophils and lung tissue from patients with COPD (isoform A: 450 vs Isoform B: 1500 relative expression (ACT)). MKK3 expression was also measured in lung samples from mice treated with lipopolysacharide (LPS) or saline. Densitometric analysis of WB data showed that LPS treated mice developed an increased total MKK3 protein expression compared to saline-treated mice (sham: 49 vs LPS: 60 A.U. p≥0.05). No significant increase in expression in these tissues was observed in mice allergic to ovalbumin. In conclusion, MKK3 is differentially expressed under non-allergic and allergic conditions.

MKK3 is a member of the p38 MAPK signaling pathway and is an important factor in non allergic inflammatory and Th1 responses. Less is known about the role of MKK3 in allergic inflammation. We investigated the role of MKK3 in murine models of non-allergic and allergic lung inflammation. Wild Type (WT) mice were instillated with lipopolysaccharide (LPS) (10 μg, i.t) or zymosan (100μg i.t.). A significant increase in neutrophil numbers in the lung were observed 24h later compared to saline controls (saline: 0.3±0.01 vs LPS: 9.4±1.9; zymosan: 6.2±0.8×10 5 /ml, n=9. p≤0.05) or MKK3 -/- KO mice (LPS: 0.36±0.04; zymosan: 0.57±0.3×10 5 /ml, n=10). WT mice also produced significant levels of IL-6, IL-12, TNF-α and INF-α as compared to saline mice. MKK3 -/- mice did not release these cytokine in response to LPS or zymosan. WT mice were sensitized twice to ovalbumin (ova, 10mg/mouse i.p in alum). From day 14 all mice were exposed to 3% ova once daily for 3 days. Lung lavages were performed 24 h after the last exposure. Exposure to ova significantly increased eosinophils number in the lungs of ova-sensitized mice as compared with sham-immunized mice (sham: 0±0 vs ova WT: 0.45±0.11, n=11; ova MKK3 -/- : 2.83±0.74, n=13×10 5 /ml). Ova-MKK3 -/- mice showed significant increase of IL-5 compared to WT ova and sham-immunized mice (sham: 0.38±0.98 vs ova WT: 3.5±1.9 and ova MKK3 -/- : 20.8±10.7 pg/ml). MKK3 -/- mice showed significantly higher levels of IgE compared to WT mice, irrespective of ova treatment (sham WT: 0±0; ova WT: 6062.3±560 pg/ml, n=6; sham MKK3 -/- : 4414.5±637, ova MKK3 -/- : 5328.8±415.8 pg/ml, n=13). In conclusion, MKK3 differentially regulates allergic and non-allergic responses in the lung.

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.