Novel molecular pathologies in asthma and COPD

Both asthma and COPD are respiratory diseases and a major global health problem with increasing prevalence. Airway inflammation is a characteristic and important hallmark in both diseases and therefore, in the past, investigations focused strongly on the immunological aspect of these disorders. In recent years, it has been shown that resident cells of the airways, in particular airway smooth muscle (ASM) cells, would be pivotal in understanding the mechanisms underlying asthma, since they are able to secrete pro-inflammatory cytokines and exert a major effector function in airway constriction. Especially the abnormal expression in ASM cells in asthmatic patients of the cell cycle regulator and pro-inflammatory gene transcription factor C/EBPα may account for many asthma-specific phenotypes (increased proliferation and increased bulk of ASM cells, increased release of inflammatory mediators). In a first phase, we analyzed the translation of the CEBPA mRNA with a translation control reporter system (TCRS), which is able to monitor translation regulation of the C/EBPα. We found an impaired translation re-initiaion in ASM cells of asthmatic patients, which coincided with decreased levels of eIF4E, an important protein for translation initiation. In a second part of this thesis, we investigated the interaction of ASM cells with house dust mite extract, a potent airborne allergen. We found that HDM extract (i) reduces C/EBPα expression in ASM cells of asthma patients, (ii) enhances the release of IL-6 and (iii) induces cell proliferation. The reduction of the C/EBPα protein is achieved trough up-regulation of calreticulin, a repressor of CEBPA mRNA translation. Therefore, the direct, not immune-mediated interaction of HDM extract with the ASM cells is able to trigger an inflammatory response in these cells and to induce an enhanced proliferation, which may finally lead to the characteristic increased muscle mass observed in the airway of asthmatic patients. These findings may be of particular importance to explain non-atopic, intrinsic asthma, which affects 30% - 50% of asthmatic subjects. In the light of these findings, new therapeutic strategies targeting regulatory mechanisms of CEBPA mRNA translation should be considered in order to restore a balanced expression of the C/EBPα protein. In a third part of this thesis, we investigated the effect of cigarette smoke on the expression levels of C/EBPα and C/EBPβ in primary lung fibroblasts. Cigarette smoke affects both C/EBPα and C/EBPβ expression via translational control mechanisms in primary lung fibroblasts. In serumfree environment, cigarette smoke increased both C/EBPα and -β expression at the translational level via the uORF mechanism. In the presence of FCS, cigarette smoke increased the levels of hnRNP E2, an inhibitor of C/EBPα translation. As a consequence, both C/EBPα and -β expression decreased with increasing concentration of cigarette smoke. In both conditions, cigarette smoke had a potent antiproliferative effect on fibroblasts. Furthermore, cigarette smoke increased the release of IL-8. We postulate that the cigarette smoke-induced imbalance of proand anti-proliferative signals provides a novel mechanism to explain many pathologies of COPD and emphysema, especially the tissue destruction defined as an imbalance between tissue injury and tissue repair. Furthermore, we showed that that the direct interaction of lung fibroblast with cigarette smoke triggers the release of pro-inflammatory mediators, contributing to the inflammatory environment that characterizes COPD

Asthma and COPD - The C/EBP Connection

Asthma and chronic obstructive pulmonary disease (COPD) are the two most prominent chronic inflammatory lung diseases with increasing prevalence. Both diseases are associated with mild or severe remodeling of the airways. In this review, we postulate that the pathologies of asthma and COPD may result from inadequate responses and/or a deregulated balance of a group of cell differentiation regulating factors, the CCAAT/Enhancer Binding Proteins (C/EBPs). In addition, we will argue that the exposure to environmental factors, such as house dust mite and cigarette smoke, changes the response of C/EBPs and are different in diseased cells. These novel insights may lead to a better understanding of the etiology of the diseases and may provide new aspects for therapies

Calreticulin Is a Negative Regulator of Bronchial Smooth Muscle Cell Proliferation

Background. Calreticulin controls the C/EBPαp42/p30 at the translational level trough a cis-regulatory CNG rich loop in the CEBPA mRNA. We determined the effects of steroids and long-acting beta-agonists on the p42/p30 ratio and on calreticulin expression in primary human bronchial smooth muscle (BSM) cells. Methods. The effects of budesonide (10−8 M) and formoterol (10−8 M) were studied in BSM cells pre-treated with siRNA targeting calreticulin. The expression of C/EBPα and calreticulin was determined by immuno-blotting. Automated cell counts were performed to measure proliferation. Results. All tested BSM cell lines (n = 5) expressed C/EBPα and calreticulin. In the presence of 5% FBS, the p42/p30 ratio significantly decreased (n = 3, P < 0.05) and coincided with BSM cell proliferation. High levels of calreticulin were associated with a decreased p42/p30 isoform ratio. FBS induced the expression of calreticulin (n = 3, P < 0.05), which was further increased by formoterol. siRNA targeting calreticulin increased the p42/p30 ratio in non-stimulated BSM cells and significantly inhibited the proliferation of PDGF-BB-stimulated BSM cells (n = 5, P < 0.05). Neither budesonide nor formoterol restored the p42 isoform expression. Conclusions. Our data show calreticulin is a negative regulator of C/EBPα protein expression in BSM cells. Modulation of calreticulin levels may provide a novel target to reduce BSM remodeling

A Prognostic Model to Predict Ruxolitinib Discontinuation and Death in Patients with Myelofibrosis

Most patients with myelofibrosis (MF) discontinue ruxolitinib (JAK1/JAK2 inhibitor) in the first 5 years of therapy due to therapy failure. As the therapeutic possibilities of MF are expanding, it is critical to identify patients predisposed to early ruxolitinib monotherapy failure and worse outcomes. We investigated predictors of early ruxolitinib discontinuation and death on therapy in 889 patients included in the "RUX-MF" retrospective study. Overall, 172 patients were alive on ruxolitinib after ≥5 years (long-term ruxolitinib, LTR), 115 patients were alive but off ruxolitinib after ≥5 yrs (short-term RUX, STR), and 123 patients died while on ruxolitinib after &lt;5 yrs (early death on ruxolitinib, EDR). The cumulative incidence of the blast phase was similar in LTR and STR patients (p = 0.08). Overall survival (OS) was significantly longer in LTR pts (p = 0.002). In multivariate analysis, PLT &lt; 100 × 109/L, Hb &lt; 10 g/dL, primary MF, absence of spleen response at 3 months and ruxolitinib starting dose &lt;10 mg BID were associated with higher probability of STR. Assigning one point to each significant variable, a prognostic model for STR (STR-PM) was built, and three groups were identified: low (score 0-1), intermediate (score 2), and high risk (score ≥ 3). The STR-PM may identify patients at higher risk of failure with ruxolitinib monotherapy who should be considered for alternative frontline strategies

Ruxolitinib in cytopenic myelofibrosis: Response, toxicity, drug discontinuation, and outcome

Background: Patients with cytopenic myelofibrosis (MF) have more limited therapeutic options and poorer prognoses compared with patients with the myeloproliferative phenotype. Aims and methods: Prognostic correlates of cytopenic phenotype were explored in 886 ruxolitinib-treated patients with primary/secondary MF (PMF/SMF) included in the RUX-MF retrospective study. Cytopenia was defined as: leukocyte count &lt;4&nbsp;×&nbsp;109 /L and/or hemoglobin &lt;11/&lt;10&nbsp;g/dL (males/females) and/or platelets &lt;100&nbsp;×&nbsp;109 /L. Results: Overall, 407 (45.9%) patients had a cytopenic MF, including 249 (52.4%) with PMF. In multivariable analysis, high molecular risk mutations (p&nbsp;=&nbsp;.04), intermediate 2/high Dynamic International Prognostic Score System (p&nbsp;&lt;&nbsp;.001) and intermediate 2/high Myelofibrosis Secondary to Polycythemia Vera and Essential Thrombocythemia Prognostic Model (p&nbsp;&lt;&nbsp;.001) remained associated with cytopenic MF in the overall cohort, PMF, and SMF, respectively. Patients with cytopenia received lower average ruxolitinib at the starting (25.2&nbsp;mg/day vs. 30.2&nbsp;mg/day, p&nbsp;&lt;&nbsp;.001) and overall doses (23.6&nbsp;mg/day vs. 26.8&nbsp;mg/day, p&nbsp;&lt;&nbsp;.001) and achieved lower rates of spleen (26.5% vs. 34.1%, p&nbsp;=&nbsp;.04) and symptom (59.8% vs. 68.8%, p&nbsp;=&nbsp;.008) responses at 6&nbsp;months compared with patients with the proliferative phenotype. Patients with cytopenia also had higher rates of thrombocytopenia at 3&nbsp;months (31.1% vs. 18.8%, p&nbsp;&lt;&nbsp;.001) but lower rates of anemia (65.6% vs. 57.7%, p&nbsp;=&nbsp;.02 at 3&nbsp;months and 56.6% vs. 23.9% at 6&nbsp;months, p&nbsp;&lt;&nbsp;.001). After competing risk analysis, the cumulative incidence of ruxolitinib discontinuation at 5&nbsp;years was 57% and 38% in patients with cytopenia and the proliferative phenotype (p&nbsp;&lt;&nbsp;.001), whereas cumulative incidence of leukemic transformation was similar (p&nbsp;=&nbsp;.06). In Cox regression analysis adjusted for Dynamic International Prognostic Score System score, survival was significantly shorter in patients with cytopenia (p&nbsp;&lt;&nbsp;.001). Conclusions: Cytopenic MF has a lower probability of therapeutic success with ruxolitinib as monotherapy and worse outcome. These patients should be considered for alternative therapeutic strategies

Proteolytic Activity Present in House-Dust-Mite Extracts Degrades ENA-78/CXCL5 and Reduces Neutrophil Migration

Background. Bronchial smooth muscle cells (BSMC) are a major source of proinflammatory and proangiogenic cytokines and chemokines, including VEGF and CXC-chemokines. CXC-chemokines act primarily on neutrophils, mediating their recruitment to and activation at the site of inflammation. In humans, house-dust mite (HDM) allergens can cause asthmatic exacerbations and trigger an inflammatory response through protease-dependent mechanisms. Objective. We investigated the effect HDM extract on the release of pro-angiogenic and proinflammatory cytokines from BSMC. Methods. Human primary BSMC were stimulated with HDM extract in the absence or presence of fetal calf serum (FCS). Twenty angiogenic cytokines were detected by a specific antibody array and modified protein levels were confirmed by ELISA. Neutrophil migration was measured using a 96-well Boyden chamber. Results. ENA-78/CXCL5 protein levels in conditioned medium of BSMC stimulated with HDM extract were significantly reduced (n=10, P<0.05) but restored in the presence of 5% FCS. HDM extracts did not affect ENA-78/CXCL5 mRNA levels. Recombinant ENA-78/CXCL5 was degraded after incubation with HDM extracts (n=7, P<0.05) but restored after the addition of the serine protease AEBSF. Neutrophil migration towards recombinant ENA-78/CXCL5 was also reduced in the presence of HDM extract. Conclusion. HDM proteases degrade ENA-78/CXCL5. Thus exposure to HDM allergens may alter ENA-78/CXCL5 levels in the lungs and may affect angiogenesis and the inflammatory response in the airways of asthma patients

Impaired translation of CCAAT/enhancer binding protein alpha mRNA in bronchial smooth muscle cells of asthmatic patients

Bronchial smooth muscle (BSM) cells of asthmatic patients have an impaired expression of CCAAT/enhancer binding protein (C/EBP) alpha, which is associated with increased proliferation