Mechanisms underlying corticosteroid resistance in patients with asthma: a review of current knowledge.

INTRODUCTION: Corticosteroids are the most cost-effective anti-inflammatory drugs available for the treatment of asthma. Despite their effectiveness, several asthmatic patients have corticosteroid resistance or insensitivity and exhibit a poor response. Corticosteroid insensitivity implies a poor prognosis due to challenges in finding alternative therapeutic options for asthma. AREAS COVERED: In this review, we describe asthma phenotypes and endotypes, as well as their differential responsiveness to corticosteroids. In addition, we describe the mechanism of action of corticosteroids underlying their regulation of the expression of glucocorticoid receptors (GRs) and their anti-inflammatory effects. Furthermore, we summarize the mechanistic evidence underlying corticosteroid-insensitive asthma, which is mainly related to changes in GR gene expression, structure, and post-transcriptional modifications. Finally, various pharmacological strategies designed to reverse corticosteroid insensitivity are discussed. EXPERT OPINION: Corticosteroid insensitivity is influenced by the asthma phenotype, endotype, and severity, and serves as an indication for biological therapy. The molecular mechanisms underlying corticosteroid-insensitive asthma have been used to develop targeted therapeutic strategies. However, the lack of clinical trials prevents the clinical application of these treatments.

Obacunone Photoprotective Effects against Solar-Simulated Radiation-Induced Molecular Modifications in Primary Keratinocytes and Full-Thickness Human Skin.

Solar radiation can cause damage to the skin, leading to various adverse effects such as sunburn, reactive oxygen species production, inflammation, DNA damage, and photoaging. To study the potential of photoprotective agents, full-thickness skin models are increasingly being used as in vitro tools. One promising approach to photoprotection involves targeting the redox-sensitive transcription factor Nrf2, which is responsible for regulating various cellular defense mechanisms, including the antioxidant response, inflammatory signaling, and DNA repair. Obacunone, a natural triterpenoid, has been identified as a potent Nrf2 agonist. The present study aims to evaluate the relevance of full-thickness (FT) skin models in photoprotection studies and to explore the potential photoprotective effects of obacunone on those models and in human keratinocytes. Phenion® full-thickness skin models and keratinocytes were incubated with increasing concentrations of obacunone and irradiated with solar-simulated radiation (SSR). Various photodamage markers were evaluated, including histological integrity, oxidative stress, apoptosis, inflammation, photoaging-related dermal markers, and photocarcinogenesis markers. Increasing doses of SSR were found to modulate various biomarkers related to sun damage in the FT skin models. However, obacunone attenuated cytotoxicity, inflammation, oxidative stress, sunburn reaction, photoaging, and photocarcinogenesis in both keratinocytes and full thickness skin models exposed to SSR. These results suggest that obacunone may have potential as a photoprotective agent for preventing the harmful effects of solar radiation on the skin.

Isocitrate dehydrogenase 2 inhibitor enasidenib synergizes daunorubicin cytotoxicity by targeting aldo-keto reductase 1C3 and ATP-binding cassette transporters.

Targeting mutations that trigger acute myeloid leukaemia (AML) has emerged as a refined therapeutic approach in recent years. Enasidenib (Idhifa) is the first selective inhibitor of mutated forms of isocitrate dehydrogenase 2 (IDH2) approved against relapsed/refractory AML. In addition to its use as monotherapy, a combination trial of enasidenib with standard intensive induction therapy (daunorubicin + cytarabine) is being evaluated. This study aimed to decipher enasidenib off-target molecular mechanisms involved in anthracycline resistance, such as reduction by carbonyl reducing enzymes (CREs) and drug efflux by ATP-binding cassette (ABC) transporters. We analysed the effect of enasidenib on daunorubicin (Daun) reduction by several recombinant CREs and different human cell lines expressing aldo-keto reductase 1C3 (AKR1C3) exogenously (HCT116) or endogenously (A549 and KG1a). Additionally, A431 cell models overexpressing ABCB1, ABCG2, or ABCC1 were employed to evaluate enasidenib modulation of Daun efflux. Furthermore, the potential synergism of enasidenib over Daun cytotoxicity was quantified amongst all the cell models. Enasidenib selectively inhibited AKR1C3-mediated inactivation of Daun in vitro and in cell lines expressing AKR1C3, as well as its extrusion by ABCB1, ABCG2, and ABCC1 transporters, thus synergizing Daun cytotoxicity to overcome resistance. This work provides in vitro evidence on enasidenib-mediated targeting of the anthracycline resistance actors AKR1C3 and ABC transporters under clinically achievable concentrations. Our findings may encourage its combination with intensive chemotherapy and even suggest that the effectiveness of enasidenib as monotherapy against AML could lie beyond the targeting of mIDH2.

Tepotinib Inhibits Several Drug Efflux Transporters and Biotransformation Enzymes: The Role in Drug-Drug Interactions and Targeting Cytostatic Resistance In Vitro and Ex Vivo.

Tepotinib is a novel tyrosine kinase inhibitor recently approved for the treatment of non-small cell lung cancer (NSCLC). In this study, we evaluated the tepotinib's potential to perpetrate pharmacokinetic drug interactions and modulate multidrug resistance (MDR). Accumulation studies showed that tepotinib potently inhibits ABCB1 and ABCG2 efflux transporters, which was confirmed by molecular docking. In addition, tepotinib inhibited several recombinant cytochrome P450 (CYP) isoforms with varying potency. In subsequent drug combination experiments, tepotinib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCB1 and ABCG2 overexpression, respectively. Remarkably, MDR-modulatory properties were confirmed in ex vivo explants derived from NSCLC patients. Furthermore, we demonstrated that anticancer effect of tepotinib is not influenced by the presence of ABC transporters associated with MDR, although monolayer transport assays designated it as ABCB1 substrate. Finally, tested drug was observed to have negligible effect on the expression of clinically relevant drug efflux transporters and CYP enzymes. In conclusion, our findings provide complex overview on the tepotinib's drug interaction profile and suggest a promising novel therapeutic strategy for future clinical investigations.

Inhibition of AKR1B10-mediated metabolism of daunorubicin as a novel off-target effect for the Bcr-Abl tyrosine kinase inhibitor dasatinib.

Bcr-Abl tyrosine kinase inhibitors significantly improved Philadelphia chromosome-positive leukaemia therapy. Apart from Bcr-Abl kinase, imatinib, dasatinib, nilotinib, bosutinib and ponatinib are known to have additional off-target effects that might contribute to their antitumoural activities. In our study, we identified aldo-keto reductase 1B10 (AKR1B10) as a novel target for dasatinib. The enzyme AKR1B10 is upregulated in several cancers and influences the metabolism of chemotherapy drugs, including anthracyclines. AKR1B10 reduces anthracyclines to alcohol metabolites that show less antineoplastic properties and tend to accumulate in cardiac tissue. In our experiments, clinically achievable concentrations of dasatinib selectively inhibited AKR1B10 both in experiments with recombinant enzyme (Ki = 0.6 µM) and in a cellular model (IC50 = 0.5 µM). Subsequently, the ability of dasatinib to attenuate AKR1B10-mediated daunorubicin (Daun) resistance was determined in AKR1B10-overexpressing cells. We have demonstrated that dasatinib can synergize with Daun in human cancer cells and enhance its therapeutic effectiveness. Taken together, our results provide new information on how dasatinib may act beyond targeting Bcr-Abl kinase, which may help to design new chemotherapy regimens, including those with anthracyclines.

ß2-adrenoreceptors control human skin microvascular reactivity.

Topical α1- and α2-adrenoreceptor (ADRA1 and 2) agonists are effective in alleviating permanent vasodilation and facial erythema associated with rosacea by inducing skin vasoconstriction. Although ß-adrenoreceptor (ADRB) antagonists are used off-label for rosacea, pharmacological and pharmacodynamic data pertaining to these receptors in skin micro-vessels are lacking. Objectives: To analyse the expression of different adrenergic receptors and their contribution to vasoreactivity in skin micro-vessels. Small arteries (500-800 µm) and arterioles (<200 µm) were studied in human foreskin tissue. Specifically, ADR-A1, -A2, -B1 and -B2 expression was assayed by immunofluorescence, polymerase chain reaction (PCR), and western blotting. Small skin artery reactivity was evaluated using ex vivo myography (500-800 µm) or a visible microscope perfusion system with precision-cut skin slices (<200 µm). ADRB2 was the most highly expressed receptor in small skin arteries and arterioles, followed by ADRA2. ADRA2 activation via brimonidine-induced vasoconstriction was greater in skin arterioles than in small skin arteries, and more potent than that with norepinephrine (NE). The use of prazosin (ADRA1 inhibitor) partially attenuated brimonidine-induced vasoconstriction, indicating some activation of ADRA1 by brimonidine, at least at 10-µM concentrations. Small skin arteries and arterioles, pre-treated with prazosin and stimulated with NE, exhibited ADRB2-mediated vasodilation, which was inhibited by the beta blockers, propranolol or timolol. This study shows that ADRB2 is predominantly expressed in small skin arteries and arterioles, and that ADRBs plays a functional role in vasodilation. The data presented here indicate that ADRBs can be a therapeutic target for the treatment of rosacea.

Selective inhibition of aldo-keto reductase 1C3: a novel mechanism involved in midostaurin and daunorubicin synergism.

Midostaurin is an FMS-like tyrosine kinase 3 receptor (FLT3) inhibitor that provides renewed hope for treating acute myeloid leukaemia (AML). The limited efficacy of this compound as a monotherapy contrasts with that of its synergistic combination with standard cytarabine and daunorubicin (Dau), suggesting a therapeutic benefit that is not driven only by FLT3 inhibition. In an AML context, the activity of the enzyme aldo-keto reductase 1C3 (AKR1C3) is a crucial factor in chemotherapy resistance, as it mediates the intracellular transformation of anthracyclines to less active hydroxy metabolites. Here, we report that midostaurin is a potent inhibitor of Dau inactivation mediated by AKR1C3 in both its recombinant form as well as during its overexpression in a transfected cell model. Likewise, in the FLT3- AML cell line KG1a, midostaurin was able to increase the cellular accumulation of Dau and significantly decrease its metabolism by AKR1C3 simultaneously. The combination of those mechanisms increased the nuclear localization of Dau, thus synergizing its cytotoxic effects on KG1a cells. Our results provide new in vitro evidence of how the therapeutic activity of midostaurin could operate beyond targeting the FLT3 receptor.

Bruton's Tyrosine Kinase Inhibitors Ibrutinib and Acalabrutinib Counteract Anthracycline Resistance in Cancer Cells Expressing AKR1C3.

Over the last few years, aldo-keto reductase family 1 member C3 (AKR1C3) has been associated with the emergence of multidrug resistance (MDR), thereby hindering chemotherapy against cancer. In particular, impaired efficacy of the gold standards of induction therapy in acute myeloid leukaemia (AML) has been correlated with AKR1C3 expression, as this enzyme metabolises several drugs including anthracyclines. Therefore, the development of selective AKR1C3 inhibitors may help to overcome chemoresistance in clinical practice. In this regard, we demonstrated that Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib efficiently prevented daunorubicin (Dau) inactivation mediated by AKR1C3 in both its recombinant form as well as during its overexpression in cancer cells. This revealed a synergistic effect of BTK inhibitors on Dau cytotoxicity in cancer cells expressing AKR1C3 both exogenously and endogenously, thus reverting anthracycline resistance in vitro. These findings suggest that BTK inhibitors have a novel off-target action, which can be exploited against leukaemia through combination regimens with standard chemotherapeutics like anthracyclines.

Targeting Pharmacokinetic Drug Resistance in Acute Myeloid Leukemia Cells with CDK4/6 Inhibitors.

Pharmacotherapy of acute myeloid leukemia (AML) remains challenging, and the disease has one of the lowest curability rates among hematological malignancies. The therapy outcomes are often compromised by the existence of a resistant AML phenotype associated with overexpression of ABCB1 and ABCG2 transporters. Because AML induction therapy frequently consists of anthracycline-like drugs, their efficiency may also be diminished by drug biotransformation via carbonyl reducing enzymes (CRE). In this study, we investigated the modulatory potential of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib on AML resistance using peripheral blood mononuclear cells (PBMC) isolated from patients with de novo diagnosed AML. We first confirmed inhibitory effect of the tested drugs on ABCB1 and ABCG2 in ABC transporter-expressing resistant HL-60 cells while also showing the ability to sensitize the cells to cytotoxic drugs even as no effect on AML-relevant CRE isoforms was observed. All tested CDK4/6 inhibitors elevated mitoxantrone accumulations in CD34+ PBMC and enhanced accumulation of mitoxantrone was found with abemaciclib and ribociclib in PBMC of FLT3-ITD- patients. Importantly, the accumulation rate in the presence of CDK4/6 inhibitors positively correlated with ABCB1 expression in CD34+ patients and led to enhanced apoptosis of PBMC in contrast to CD34- samples. In summary, combination therapy involving CDK4/6 inhibitors could favorably target multidrug resistance, especially when personalized based on CD34- and ABCB1-related markers.

Entrectinib reverses cytostatic resistance through the inhibition of ABCB1 efflux transporter, but not the CYP3A4 drug-metabolizing enzyme.

Entrectinib is a new tyrosine kinase inhibitor that was recently approved for the treatment of ROS1-positive metastatic non-small cell lung cancer (NSCLC). In this study, we aimed to characterize its potential to act as a modulator of pharmacokinetic cytostatic resistance and perpetrator of drug interactions. In accumulation studies, entrectinib exhibited potent inhibition of ABCB1, while only moderate interaction was recorded for ABCG2 and ABCC1 efflux transporters. Furthermore, incubation assays revealed the potential of this drug to inhibit various recombinant cytochrome P450 enzymes, which can be ranked according to inhibitory affinities as follows: CYP2C8 ≈ CYP3A4 > CYP2C9 > CYP2C19 ≈ CYP3A5 > CYP2D6 > CYP2B6 > CYP1A2. Additionally, in silico docking analysis confirmed entrectinib's interactions with ABCB1 and CYP3A4 and resolved their possible molecular background. In subsequent drug combination experiments, we demonstrated the ability of entrectinib to synergize with daunorubicin in various ABCB1-expressing cellular models. Moreover, the comparative proliferation study results suggested that the anticancer efficacy of entrectinib is not affected by the functional presence of tested ABC transporters. In contrast to ABCB1-related data, no resistance reversal effect was recorded for the combination with docetaxel in HepG2-CYP3A4 cells. In the final experimental set, we observed no significant changes in ABCB1, ABCG2, ABCC1 or CYP3A4 gene expression in NSCLC cells exposed to entrectinib. In summary, our work indicates that entrectinib may be a perpetrator of clinically relevant pharmacokinetic drug interactions and modulator of ABCB1-mediated resistance. Our in vitro results might provide a valuable foundation for future clinical investigations.

Interactions of antileukemic drugs with daunorubicin reductases: could reductases affect the clinical efficacy of daunorubicin chemoregimens?

Although novel anticancer drugs are being developed intensively, anthracyclines remain the gold standard in the treatment of acute myeloid leukaemia (AML). The reductive conversion of daunorubicin (Dau) to less active daunorubicinol (Dau-ol) is an important mechanism that contributes to the development of pharmacokinetic anthracycline resistance. Dau is a key component in many AML regimes, in which it is combined with many drugs, including all-trans-retinoic acid (ATRA), cytarabine, cladribine and prednisolone. In the present study, we investigated the influence of these anticancer drugs on the reductive Dau metabolism mediated by the aldo-keto reductases AKR1A1, 1B10, 1C3, and 7A2 and carbonyl reductase 1 (CBR1). In incubation experiments with recombinant enzymes, cladribine and cytarabine did not significantly inhibit the activity of the tested enzymes. Prednisolone inhibited AKR1C3 with an IC50 of 41.73 µM, while ATRA decreased the activity of AKR1B10 (IC50 = 78.33 µM) and AKR1C3 (IC50 = 1.17 µM). Subsequent studies showed that AKR1C3 inhibition mediated by ATRA exhibited tight binding (Kiapp = 0.54 µM). Further, the combination of 1 µM ATRA with different concentrations of Dau demonstrated synergistic effects in HCT116 and KG1a human cells expressing AKR1C3. Our results suggest that ATRA-mediated inhibition of AKR1C3 can contribute to the mechanisms that are hidden beyond the beneficial clinical outcome of the ATRA-Dau combination.

Cyclin-dependent kinase inhibitors AZD5438 and R547 show potential for enhancing efficacy of daunorubicin-based anticancer therapy: Interaction with carbonyl-reducing enzymes and ABC transporters.

Daunorubicin (DAUN) has served as an anticancer drug in chemotherapy regimens for decades and is still irreplaceable in treatment of acute leukemias. The therapeutic outcome of DAUN-based therapy is compromised by its cardiotoxicity and emergence of drug resistance. This phenomenon is often caused by pharmacokinetic mechanisms such as efflux of DAUN from cancer cells through ATP-binding cassette (ABC) transporters and its conversion to less cytostatic but more cardiotoxic daunorubicinol (DAUN-OL) by carbonyl reducing enzymes (CREs). Here we aimed to investigate, whether two cyclin-dependent kinase inhibitors, AZD5438 and R547, can interact with these pharmacokinetic mechanisms and reverse DAUN resistance. Using accumulation assays, we revealed AZD5438 as potent inhibitor of ABCC1 showing also weaker inhibitory effect to ABCB1 and ABCG2. Combination index analysis, however, shown that inhibition of ABCC1 does not significantly contribute to synergism between AZD5438 and DAUN in MDCKII-ABCC1 cells, suggesting predominant role of other mechanism. Using pure recombinant enzymes, we found both tested drugs to inhibit CREs with aldo-keto reductase 1C3 (AKR1C3). This interaction was further confirmed in transfected HCT-116 cells. Moreover, these cells were sensitized to DAUN by both compounds as Chou-Talalay combination index analysis showed synergism in AKR1C3 transfected HCT-116, but not in empty vector transfected control cell line. In conclusion, we propose AZD5438 and R547 as modulators of DAUN resistance that can prevent AKR1C3-mediated DAUN biotransformation to DAUN-OL. This interaction could be beneficially exploited to prevent failure of DAUN-based therapy as well as the undesirable cardiotoxic effect of DAUN-OL.

Buparlisib is a novel inhibitor of daunorubicin reduction mediated by aldo-keto reductase 1C3.

Buparlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor and is currently under clinical evaluation for the treatment of different cancers. Because PI3K signalling is related to cell proliferation and resistance to chemotherapy, new therapeutic approaches are focused on combining PI3K inhibitors with other anti-cancer therapeutics. Carbonyl-reducing enzymes catalyse metabolic detoxification of anthracyclines and reduce their cytotoxicity. In the present work, the effects of buparlisib were tested on six human recombinant carbonyl-reducing enzymes: AKR1A1, AKR1B1, AKR1B10, AKR1C3, and AKR7A2 from the aldo-keto reductase superfamily and CBR1 from the short-chain dehydrogenase/reductase superfamily, all of which participate in the metabolism of daunorubicin. Buparlisib exhibited the strongest inhibitory effect on recombinant AKR1C3, with a half-maximal inhibitory concentration (IC50) of 9.5 µM. Its inhibition constant Ki was found to be 14.0 µM, and the inhibition data best fitted a mixed-type mode with α = 0.6. The same extent of inhibition was observed at the cellular level in the human colorectal carcinoma HCT 116 cell line transfected with a plasmid encoding the AKR1C3 transcript (IC50 = 7.9 µM). Furthermore, we performed an analysis of flexible docking between buparlisib and AKR1C3 and found that buparlisib probably occupies a part of the binding site for a cofactor most likely via the trifluoromethyl group of buparlisib interacting with catalytic residue Tyr55. In conclusion, our results show a novel PI3K-independent effect of buparlisib that may improve therapeutic efficacy and safety of daunorubicin by preventing its metabolism by AKR1C3.

Mucin 1 deficiency mediates corticosteroid insensitivity in asthma.

BACKGROUND: The loss of corticosteroid efficacy is an important issue in severe asthma management and may lead to poor asthma control and deterioration of airflow. Recent data indicate that Mucin 1 (MUC1) membrane mucin can mediate corticosteroid efficacy in chronic rhinosinusitis, but the role of MUC1 in uncontrolled severe asthma is unknown. The objective was to analyze the previously unexplored role of MUC1 on corticosteroid efficacy in asthma. METHODS: Mucin 1 expression was evaluated by real-time PCR in human bronchial epithelial cells (HBEC) and blood neutrophils from uncontrolled severe asthma (n = 27), controlled mild asthma (n = 16), and healthy subjects (n = 13). IL-8, MMP9, and GM-CSF were measured by ELISA in HBEC and neutrophils. An asthma model of ovalbumin (OVA) was used in MUC1 KO and WT C57BL/6 mice according to ARRIVE guidelines. RESULTS: Mucin 1-CT expression was downregulated in bronchial epithelial cells and peripheral blood neutrophils from severe asthma patients compared with mild asthma and healthy subjects (P < 0.05). Daily dose of inhaled corticosteroids (ICS) inversely correlated with MUC1 expression in neutrophils from mild and severe asthma (ρ = -0.71; P < 0.0001). Dexamethasone showed lower anti-inflammatory effects in severe asthma peripheral blood neutrophils and HBECs stimulated with lipopolysaccharide (LPS) than in cells from mild asthma. Glucocorticoid receptor (GR)-α phosphorylated at serine 226 was increased in cells from severe asthma, and the MUC1-CT/GRα complex was downregulated in severe asthma cells. OVA asthma model in MUC1 KO mice was resistant to the anti-inflammatory effects of dexamethasone. CONCLUSION: Mucin 1-CT modulates corticosteroid efficacy in vitro and in vivo asthma models.

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study.

BACKGROUND: Pulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation (V)/perfusion (Q) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown. OBJECTIVE: The study of JAK2 as potential target to treat PH in IPF. METHODS AND RESULTS: JAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; P<0.0001) compared with PA from control subjects (n=10). PA remodelling was evaluated in human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs) from patients with IPF in vitro treated with the JAK2 inhibitor JSI-124 or siRNA-JAK2 and stimulated with transforming growth factor beta. Both JSI-124 and siRNA-JAK2 inhibited the HPAEC to mesenchymal transition and the HPASMCs to myofibroblast transition and proliferation. JAK2 inhibition induced small PA relaxation in precision-cut lung slice experiments. PA relaxation was dependent of the large conductance calcium-activated potassium channel (BKCa). JAK2 inhibition activated BKCa channels and reduced intracellular Ca2+. JSI-124 1 mg/kg/day, reduced bleomycin-induced lung fibrosis, PA remodelling, right ventricular hypertrophy, PA hypertension and V/Q mismatching in rats. The animal studies followed the ARRIVE guidelines. CONCLUSIONS: JAK2 participates in PA remodelling and tension and may be an attractive target to treat IPF associated to PH.

The JAK2 pathway is activated in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal fibrotic disorder, with no curative therapies. The signal transducer and activator of transcription 3 (STAT3) protein is activated in lung fibroblasts and alveolar type II cells (ATII), thereby contributing to lung fibrosis in IPF. Although activation of Janus kinase 2 (JAK2) has been implicated in proliferative disorders, its role in IPF is unknown. The aim of this study was to analyze JAK2 activation in IPF, and to determine whether JAK2/STAT3 inhibition is a potential therapeutic strategy for this disease. METHODS AND RESULTS: JAK2/p-JAK2 and STAT3/pSTAT3 expression was evaluated using quantitative real time-PCR, western blotting, and immunohistochemistry. Compared to human healthy lung tissue (n = 10) both proteins were upregulated in the lung tissue of IPF patients (n = 12). Stimulating primary ATII and lung fibroblasts with transforming growth factor beta 1 or interleukin (IL)-6/IL-13 activated JAK2 and STAT3, inducing epithelial to mesenchymal and fibroblast to myofibroblast transitions. Dual p-JAK2/p-STAT3 inhibition with JSI-124 or silencing of JAK2 and STAT3 genes suppressed ATII and the fibroblast to myofibroblast transition, with greater effects than the sum of those obtained using JAK2 or STAT3 inhibitors individually. Dual rather than single inhibition was also more effective for inhibiting fibroblast migration, preventing increases in fibroblast senescence and Bcl-2 expression, and ameliorating impaired autophagy. In rats administered JSI-124, a dual inhibitor of p-JAK2/p-STAT3, at a dose of 1 mg/kg/day, bleomycin-induced lung fibrosis was reduced and collagen deposition in the lung was inhibited, as were JAK2 and STAT3 activation and several markers of fibrosis, autophagy, senescence, and anti-apoptosis. CONCLUSIONS: JAK2 and STAT3 are activated in IPF, and their dual inhibition may be an attractive strategy for treating this disease.

MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Current evidence suggests that membrane-tethered mucins could mediate corticosteroid efficacy, interacting with glucocorticoid receptor (GR) in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Mucin 4 (MUC4)-tethered mucin is expressed in nasal polyp (NP) epithelial cells and upregulated under inflammatory conditions. Moreover, MUC4ß has the capacity to interact with other intracellular proteins. We hypothesized that MUC4 modulates corticosteroid efficacy of patients with CRSwNP. OBJECTIVE: We sought to analyze the role of MUC4 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. METHODS: Eighty-one patients with CRSwNP took oral corticosteroids for 15 days. Corticosteroid resistance was evaluated by using nasal endoscopy. Expression of MUC4 and MUC4ß was evaluated by means of real-time PCR, Western blotting, and immunohistochemistry. BEAS-2B knockdown with RNA interference for MUC4 (small interfering RNA [siRNA]-MUC4) was used to analyze the role of MUC4 in the anti-inflammatory effects of dexamethasone. RESULTS: Twenty-two patients had NPs resistant to oral corticosteroids. MUC4 expression was upregulated in these patients. In siRNA-MUC4 BEAS-2B airway epithelial cells dexamethasone produced higher anti-inflammatory effects, increased inhibition of phospho-extracellular signal-regulated kinase 1/2, increased mitogen-activated protein kinase phosphatase 1 expression, and increased glucocorticoid response element activation. Immunoprecipitation and immunofluorescence experiments revealed that MUC4ß forms a complex with GRα in the nuclei of NP epithelial cells from corticosteroid-resistant patients. CONCLUSION: MUC4ß participates in the corticosteroid resistance process, inhibiting normal GRα nuclear function. The high expression of MUC4 in patients with CRSwNP might participate in corticosteroid resistance.

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by chronic pulmonary inflammation punctuated by periods of viral exacerbations. Recent evidence suggests that the combination of roflumilast with corticosteroids may improve the compromised anti-inflammatory properties of corticosteroids in COPD. We analyzed differential and combination anti-inflammatory effects of dexamethasone and roflumilast N-oxide in human bronchial epithelial cells (HBECs) stimulated with viral toll like receptor (TLR) agonists. METHODS: Lung tissue and HBECs were isolated from healthy (n = 15), smokers (n = 12) and smokers with COPD (15). TLR3 expression was measured in lung tissue and in HBECs. IL-8 secretion was measured in cell cultures after TLR3 stimulation with poly I:C 10 µg/mL. RESULTS: We found that TLR3 expression was increased by 1.95 fold (protein) and 2.5 fold (mRNA) in lung tissues from smokers with COPD and inversely correlated with lung function. The TLR3 agonist poly I:C 10 µg/mL increased the IL-8 release in HBECs that was poorly inhibited by dexamethasone in smokers (24.5%) and smokers with COPD (21.6%). In contrast, roflumilast showed similar inhibitory effects on IL-8 release in healthy (58.8%), smokers (56.6%) and smokers with COPD (50.5%). The combination of roflumilast N-oxide and dexamethasone showed additive inhibitory effects. Mechanistically, roflumilast N-oxide when combined with dexamethasone increased the expression of MKP1, and enhanced the inhibitory effects on phospho-p38, AP1 and NFκB activities which may explain the additive anti-inflammatory effects. CONCLUSIONS: Altogether, our data provide in vitro evidence for a possible clinical utility to add roflumilast on top of inhaled corticosteroid in COPD.

Mucin 1 downregulation associates with corticosteroid resistance in chronic rhinosinusitis with nasal polyps.

BACKGROUND: A number of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to oral corticosteroids. Mucin 1 (MUC1) shows anti-inflammatory properties, and its cytoplasmic tail (CT) interacts with transcription factors, facilitating their nuclear translocation. Because glucocorticoid receptor (GR) nuclear translocation is key to the anti-inflammatory effect of corticosteroids, we hypothesized that MUC1 is involved in the effectiveness of corticosteroids. OBJECTIVE: To analyze the role of MUC1 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. METHODS: Seventy-three patients with CRSwNP took oral corticosteroids for 15 days. Corticosteroid resistance was evaluated by nasal endoscopy. The expression of MUC1 and MUC1 CT was evaluated by real-time PCR, Western blotting, and immunohistochemistry. Beas-2B knockdown with RNA interference for MUC1 (siRNA-MUC1) was used to analyze the role of MUC1 in the anti-inflammatory effects of dexamethasone. RESULTS: Nineteen patients had nasal polyps that were resistant to oral corticosteroids (NP-CR). MUC1 expression was downregulated in these patients. Primary epithelial cells from patients with NP-CR were insensitive to the anti-inflammatory effects of dexamethasone. In siRNA-MUC1 Beas-2B, dexamethasone showed weaker anti-inflammatory effects, a reduced inhibition of phospho-extracellular-signal-regulated kinases 1/2, a less severe mitogen-activated protein kinase phosphatase 1 increase, and a reduced GR nuclear translocation. Immunoprecipitation experiments revealed that MUC1-CT and GRα form protein complexes and translocate to the nucleus in response to dexamethasone. MUC1-CT-GRα complex was downregulated in NP-CR tissue. CONCLUSION: MUC1-CT participates in the corticosteroid response that mediates GRα nuclear translocation. The low expression of MUC1 in patients with CRSwNP may participate in corticosteroid resistance.