Phosphodiesterase-4 Inhibitor Roflumilast Research Articles

Roflumilast ameliorates GAN diet-induced non-alcoholic fatty liver disease by reducing hepatic steatosis and fibrosis in ob/ob mice

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent progressive liver disease. Currently, there is only one drug for NAFLD treatment, and the options are limited. Phosphodiesterase-4 (PDE-4) inhibitors have potential in treating NAFLD. Therefore, this study aims to investigate the effect of roflumilast on NAFLD. Here, we fed ob/ob mice to induce the NAFLD model by GAN diet. Roflumilast (1 mg/kg) was administered orally once daily. Semaglutide (20 nmol/kg), used as a positive control, was injected subcutaneously once daily. Our findings showed that roflumilast has beneficial effects on NAFLD. Roflumilast prevented body weight gain and improved lipid metabolism in ob/ob-GAN NAFLD mice. In addition, roflumilast decreased hepatic steatosis by down-regulating the expression of hepatic fatty acid synthesis genes (SREBP1c, FASN, and CD36) and improving oxidative stress. Roflumilast not only reduced liver injury by decreasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, but also ameliorated hepatic inflammation by reducing the gene expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). Roflumilast lessened liver fibrosis by inhibiting the expression of fibrosis mRNA (TGFβ1, α-SMA, COL1a1, and TIMP-1). Collectively, roflumilast could ameliorate NAFLD, especially in reducing hepatic steatosis and fibrosis. Our findings suggested a PDE-4 inhibitor roflumilast could be a potential drug for NAFLD.

Differential effects of two phosphodiesterase 4 inhibitors against lipopolysaccharide-induced neuroinflammation in mice

BackgroundSeveral phosphodiesterase 4 (PDE4) inhibitors have emerged as potential therapeutics for central nervous system (CNS) diseases. This study investigated the pharmacological effects of two selective PDE4 inhibitors, roflumilast and zatolmilast, against lipopolysaccharide-induced neuroinflammation.ResultsIn BV-2 cells, the PDE4 inhibitor roflumilast reduced the production of nitric oxide and tumor necrosis factor-α (TNF-α) by inhibiting NF-κB phosphorylation. Moreover, mice administered roflumilast had significantly reduced TNF-α, interleukin-1β (IL-1β), and IL-6 levels in plasma and brain tissues. By contrast, zatolmilast, a PDE4D inhibitor, showed no anti-neuroinflammatory effects in vitro or in vivo. Next, in vitro and in vivo pharmacokinetic studies of these compounds in the brain were performed. The apparent permeability coefficients of 3 µM roflumilast and zatolmilast were high (> 23 × 10–6 cm/s) and moderate (3.72–7.18 × 10–6 cm/s), respectively, and increased in a concentration-dependent manner in the MDR1-MDCK monolayer. The efflux ratios were < 1.92, suggesting that these compounds are not P-glycoprotein substrates. Following oral administration, both roflumilast and zatolmilast were slowly absorbed and eliminated, with time-to-peak drug concentrations of 2–2.3 h and terminal half-lives of 7–20 h. Assessment of their brain dispositions revealed the unbound brain-to-plasma partition coefficients of roflumilast and zatolmilast to be 0.17 and 0.18, respectively.ConclusionsThese findings suggest that roflumilast, but not zatolmilast, has the potential for use as a therapeutic agent against neuroinflammatory diseases.

Phosphodiesterase (PDE) 4 inhibition boosts Schwann cell myelination in a 3D regeneration model

Phosphodiesterase 4 (PDE4) inhibitors have been extensively researched for their anti-inflammatory and neuroregenerative properties. Despite the known neuroplastic and myelin regenerative properties of nonselective PDE4 inhibitors on the central nervous system, the direct impact on peripheral remyelination and subsequent neuroregeneration has not yet been investigated. Therefore, to examine the possible therapeutic effect of PDE4 inhibition on peripheral glia, we assessed the differentiation of primary rat Schwann cells exposed in vitro to the PDE4 inhibitor roflumilast. To further investigate the differentiation promoting effects of roflumilast, we developed a 3D model of rat Schwann cell myelination that closely resembles the in vivo situation. Using these in vitro models, we demonstrated that pan-PDE4 inhibition using roflumilast significantly promoted differentiation of Schwann cells towards a myelinating phenotype, as indicated by the upregulation of myelin proteins, including MBP and MAG. Additionally, we created a unique regenerative model comprised of a 3D co-culture of rat Schwann cells and human iPSC-derived neurons. Schwann cells treated with roflumilast enhanced axonal outgrowth of iPSC-derived nociceptive neurons, which was accompanied by an accelerated myelination speed, thereby showing not only phenotypic but also functional changes of roflumilast-treated Schwann cells. Taken together, the PDE4 inhibitor roflumilast possesses a therapeutic benefit to stimulate Schwann cell differentiation and, subsequently myelination, as demonstrated in the biologically relevant in vitro platform used in this study. These results can aid in the development of novel PDE4 inhibition-based therapies in the advancement of peripheral regenerative medicine.

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS.

Recovering object-location memories after sleep deprivation-induced amnesia

It is well established that sleep deprivation after learning impairs hippocampal memory processes and can cause amnesia. It is unknown, however, whether sleep deprivation leads to the loss of information or merely the suboptimal storage of information that is difficult to retrieve. Here, we show that hippocampal object-location memories formed under sleep deprivation conditions can be successfully retrieved multiple days following training, using optogenetic dentate gyrus (DG) memory engram activation or treatment with the clinically approved phosphodiesterase 4 (PDE4) inhibitor roflumilast. Moreover, the combination of optogenetic DG memory engram activation and roflumilast treatment, 2days following training and sleep deprivation, made the memory more persistently accessible for retrieval even several days later (i.e., without further optogenetic or pharmacological manipulation). Altogether, our studies in mice demonstrate that sleep deprivation does not necessarily cause memory loss but instead leads to the suboptimal storage of information that cannot be retrieved without drug treatment or optogenetic stimulation. Furthermore, our findings suggest that object-location memories, consolidated under sleep deprivation conditions and thought to be lost, can be made accessible again several days after the learning and sleep deprivation episode, using the clinically approved PDE4 inhibitor roflumilast.

Roflumilast-Mediated Phosphodiesterase 4D Inhibition Reverses Diabetes-Associated Cardiac Dysfunction and Remodeling: Effects Beyond Glucose Lowering

Patients with type 2 diabetes have a substantial risk of developing cardiovascular disease. Phosphodiesterase 4 (PDE4) dysregulation is of pathophysiological importance in metabolic disorders. For determination of the role of PDE4 in diabetic cardiac dysfunction, mice fed with a high-fat diet (HFD) were treated by pharmacological inhibition of PDE4 or cardiac specific knocking down of PDE4D. Mice on HFD developed diabetes and cardiac dysfunction with increased cardiac PDE4D5 expression. PDE4 inhibitor roflumilast can reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of PDE4D expression and increase of muscle specific miRNA miR-1 level in hearts. Either cardiac specific PDE4D knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD mice, despite persistence of hyperglycemia. Findings of gain- and loss-of-function studies of PDE4D in cardiomyocytes indicated that inhibition of insulin-induced PDE4D protected cardiac hypertrophy by preserving miR-1 expression in cardiomyocytes through promoting cAMP-CREB-Sirt1 signaling-induced SERCA2a expression. We further revealed that insulin also induced PDE4D expression in cardiac fibroblasts, which causes cardiac fibrosis through TGF-β1 signaling-mediated miR-1 reduction. Importantly, the expression of PDE4D5 was increased in human failing hearts of individuals with diabetes. These studies elucidate a novel mechanism by which hyperinsulinemia-induced cardiac PDE4D expression contributes to diabetic cardiac remodeling through reducing the expression of miR-1 and upregulation of miR-1 target hypertrophy and fibrosis-associated genes. Our study suggests a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction in diabetes in addition to lowering glucose.

Overexpression of PDE4D in mouse liver is sufficient to trigger NAFLD and hypertension in a CD36-TGF-β1 pathway: therapeutic role of roflumilast

Emerging evidence has shown that nonalcoholic fatty liver disease (NAFLD) may be both a consequence and a cause of hypertension. Recent studies have demonstrated that phosphodiesterase 4 (PDE4)-cAMP signaling represents a pathway relevant to the pathophysiology of metabolic disorders. This study aims to investigate the impact and the underlying mechanism of PDE4 in the pathogenesis of NAFLD and its associated hypertension. Here we demonstrated that high-fat-diet (HFD) fed mice developed NAFLD and hypertension, with an associated increase in hepatic PDE4D expression, which can be prevented and even reversed by PDE4 inhibitor roflumilast. Furthermore, we demonstrated that hepatic overexpression of PDE4D drove significant hepatic steatosis and elevation of blood pressure. Mechanistically, PDE4D activated fatty acid translocase CD36 signaling which facilitates hepatic lipid deposition, resulting in TGF-β1 production by hepatocytes and excessive TGF-β1 signaling in vessels and consequent hypertension. Specific silencing of TGF-β1 in hepatocytes by siRNA using poly (β-amino ester) nanoparticles significantly normalized hepatic PDE4D overexpression-activated TGF-β1 signaling in vessels and hypertension. Together, the conclusions indicated that PDE4D plays an important role in the pathogenesis of NAFLD and associated hypertension via activation of CD36-TGF-β1 signaling in the liver. PDE4 inhibitor such as roflumilast, which is clinically approved for chronic obstructive pulmonary disease (COPD) treatment, has the potential to be used as a preventive or therapeutic drug against NAFLD and associated hypertension in the future.

Tanimilast, A Novel Inhaled Pde4 Inhibitor for the Treatment of Asthma and Chronic Obstructive Pulmonary Disease.

Chronic respiratory diseases are the third leading cause of death, behind cardiovascular diseases and cancer, affecting approximately 550 million of people all over the world. Most of the chronic respiratory diseases are attributable to asthma and chronic obstructive pulmonary disease (COPD) with this latter being the major cause of deaths. Despite differences in etiology and symptoms, a common feature of asthma and COPD is an underlying degree of airways inflammation. The nature and severity of this inflammation might differ between and within different respiratory conditions and pharmacological anti-inflammatory treatments are unlikely to be effective in all patients. A precision medicine approach is needed to selectively target patients to increase the chance of therapeutic success. Inhibitors of the phosphodiesterase 4 (PDE4) enzyme like the oral PDE4 inhibitor roflumilast have shown a potential to reduce inflammatory-mediated processes and the frequency of exacerbations in certain groups of COPD patients with a chronic bronchitis phenotype. However, roflumilast use is dampened by class related side effects as nausea, diarrhea, weight loss and abdominal pain, resulting in both substantial treatment discontinuation in clinical practice and withdrawal from clinical trials. This has prompted the search for PDE4 inhibitors to be given by inhalation to reduce the systemic exposure (and thus optimize the systemic safety) and maximize the therapeutic effect in the lung. Tanimilast (international non-proprietary name of CHF6001) is a novel highly potent and selective inhaled PDE4 inhibitor with proven anti-inflammatory properties in various inflammatory cells, including leukocytes derived from asthma and COPD patients, as well as in experimental rodent models of pulmonary inflammation. Inhaled tanimilast has reached phase III clinical development by showing promising pharmacodynamic results associated with a good tolerability and safety profile, with no evidence of PDE4 inhibitors class-related side effects. In this review we will discuss the main outcomes of preclinical and clinical studies conducted during tanimilast development, with particular emphasis on the characterization of the pharmacodynamic profile that led to the identification of target populations with increased therapeutic potential in inflammatory respiratory diseases.

Positive effects of roflumilast on behavior, neuroinflammation, and white matter injury in mice with global cerebral ischemia.

Inhibition of phosphodiesterase 4 (PDE4) is a promising pharmacological strategy for the treatment of cerebral ischemic conditions. To increase the relevance and increase the translational value of preclinical studies, it is important to conduct experiments using different animal species and strains, different animal models, and to evaluate long-term functional outcomes after cerebral ischemia. In the present study, the effects of the selective PDE4 inhibitor roflumilast were evaluated in vivo and in vitro. Balb/c mice were subjected to bilateral common carotid artery occlusion (BCCAO) and tested during 21 days in multiple behavioral tasks to investigate the long-term effects of roflumilast on functional recovery. The effects of roflumilast were also investigated on hippocampal cell loss, white matter injury, and expression of neuroinflammatory markers. Roflumilast prevented cognitive and emotional deficits induced by BCCAO in mice. Roflumilast also prevented neurodegeneration and reduced the white matter damage in the brain of ischemic animals. Besides, roflumilast decreased Iba-1 (microglia marker) levels and increased Arginase-1 (Arg-1; microglia M2 phenotype marker) levels in the hippocampus of these mice. Likewise, roflumilast suppressed inducible nitric oxide synthase (microglia M1 phenotype marker) expression and increased Arg-1 levels in a primary mouse microglia culture. These findings support evidence that PDE4 inhibition by roflumilast might be beneficial in cerebral ischemic conditions. The neuroprotective effects of roflumilast appear to be mediated by a decrease in neuroinflammation.

Roflumilast protects against spatial memory impairments and exerts anti-inflammatory effects after transient global cerebral ischemia.

Phosphodiesterase 4 (PDE4) inhibitors have been shown to present beneficial effects in cerebral ischemic injury because of their ability to improve cognition and target different phases and mechanisms of cerebral ischemia, including apoptosis, neurogenesis, angiogenesis, and inflammation. The present study investigated whether repeated treatment with the PDE4 inhibitor roflumilast rescued memory loss and attenuated neuroinflammation in rats following transient global cerebral ischemia (TGCI). TGCI caused memory impairments, neuronal loss (reflected by Neuronal nuclei (NeuN) immunoreactivity), and compensatory neurogenesis (reflected by doublecortin (DCX) immunoreactivity) in the hippocampus. Also, increases in the protein expression of the phosphorylated response element-binding protein (pCREB) and inflammatory markers such as the glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba-1), were detected in the hippocampus in TGCI rats. Repeated treatment with roflumilast (0.003 and 0.01mg/kg) prevented spatial memory deficits without promoting hippocampal protection in ischemic animals. Roflumilast increased the levels of pCREB, arginase-1, interleukin (IL) 4, and IL-10 in the hippocampus 21days after TGCI. These data suggest a protective effect of roflumilast against functional sequelae of cerebral ischemia, which might be related to its anti-inflammatory properties.

Phosphodiesterase 4 Inhibitors in Allergic Rhinitis/Rhinosinusitis.

Allergic rhinitis/rhinosinusitis (AR) is the most common allergic disease. It affects patients’ quality of life and may influence the severity of lower airway disease such as asthma. Therefore, its treatment is of great importance. AR is treated by a combination of effective approaches; however, in some patients, the disease is uncontrolled. In the last several years, the concept of AR has shifted from increased T helper 2 (Th2) cell signaling and downstream inflammation to disease phenotypes with non-Th2-mediated inflammation. AR is a largely heterogenous group of airway diseases, and as such, research should not only focus on immunosuppressive agents (e.g., corticosteroids) but should also include targeted immunomodulatory pathways. Here, we provide an overview of novel therapies, focusing on the role of phosphodiesterase-4 (PDE4) inhibitors in AR. PDE4 inhibitors are potent anti-inflammatory agents that are used for the treatment of inflammatory airway diseases including AR. The PDE4 inhibitor roflumilast was shown to effectively control symptoms of AR in a randomized, placebo-controlled, double-blinded, crossover study in patients with a history of AR. However, only a few PDE4 inhibitors have proceeded to phase II and III clinical trials, due to insufficient clinical efficacy and adverse effects. Research is ongoing to develop more effective compounds with fewer side effects that target specific inflammatory pathways in disease pathogenesis and can provide more consistent benefit to patients with upper airway allergic diseases. Novel specific PDE4 inhibitors seem to fulfill these criteria.

The Phosphodiesterase-4 Inhibitor Roflumilast, a Potential Treatment for the Comorbidity of Memory Loss and Depression in Alzheimer's Disease: A Preclinical Study in APP/PS1 Transgenic Mice.

BackgroundDepression is highly related to Alzheimer’s disease (AD), yet no effective treatment is available. Phosphodiesterase-4 (PDE4) has been considered a promising target for treatment of AD and depression. Roflumilast, the first PDE4 inhibitor approved for clinical use, improves cognition at doses that do not cause side effects such as emesis.MethodsHere we examined the effects of roflumilast on behavioral dysfunction and the related mechanisms in APPswe/PS1dE9 transgenic mice, a widely used model of AD. Mice at 10 months of age were examined for memory in the novel object recognition and Morris water-maze tests and depression-like behavior in the tail-suspension test and forced swimming test before killing for neurochemical assays.ResultsIn the novel object recognition and Morris water-maze, APPswe/PS1dE9 mice showed significant cognitive declines, which were reversed by roflumilast at 5 and 10 mg/kg orally once per day. In the tail-suspension test and forced swimming test, the AD mice showed prolonged immobility time, which was also reversed by roflumilast. In addition, the staining of hematoxylin–eosin and Nissl showed that roflumilast relieved the neuronal cell injuries, while terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling analysis indicated that roflumilast ameliorated cell apoptosis in AD mice. Further, roflumilast reversed the decreased ratio of B-cell lymphoma-2/Bcl-2-associated X protein and the increased expression of PDE4B and PDE4D in the cerebral cortex and hippocampus of AD mice. Finally, roflumilast reversed the decreased levels of cyclic AMP (cAMP) and expression of phosphorylated cAMP response element-binding protein and brain derived neurotrophic factor in AD mice.ConclusionsTogether, these results suggest that roflumilast not only improves learning and memory but also attenuates depression-like behavior in AD mice, likely via PDE4B/PDE4D-mediated cAMP/cAMP response element-binding protein/brain derived neurotrophic factor signaling. Roflumilast can be a therapeutic agent for AD, in particular the comorbidity of memory loss and depression.

Persistence of the extinction of fear memory requires late-phase cAMP/PKA signaling in the infralimbic cortex.

Fear extinction is a form of new learning that inhibits expression of the original fear memory without erasing the conditioned stimulus-unconditioned stimulus association. Much is known about the mechanisms that underlie the acquisition of extinction, but the way in which fear extinction is maintained has been scarcely explored. Evidence suggests that protein kinase A (PKA) in the frontal cortex might be related to the persistence of extinction. Phosphodiesterase-4 (PDE4) specifically hydrolyzes cyclic adenosine monophosphate (cAMP). The present study evaluated the effect of the selective PDE4 inhibitor roflumilast (ROF; 0.01, 0.03, and 0.1mg/kg given i.p.) on acquisition and consolidation of the extinction of fear memory in male Wistar rats in a contextual fear conditioning paradigm. When administered before acquisition, 0.1mg/kg ROF disrupted short-term (1day) extinction recall. In contrast, 0.03mg/kg ROF administration in the late consolidation phase (3h after extinction learning) but not in the early phase immediately after learning improved long-term extinction recall at 11days, suggesting potentiation of the persistence of extinction. This effect of ROF requires the first (day 1) exposure to the context. A similar effect was observed when 9ng ROF or 30µM 8-bromoadenosine 3',5'-cAMP (PKA activator) was directly infused in the infralimbic cortex (IL), a brain region necessary for memory extinction. The PKA activity-dependent ROF-induced effect in the IL was correlated with an increase in its brain-derived neurotrophic factor (BDNF) protein expression, while blockade of PKA with 10µM H89 in the IL abolished the ROF-induced increase in BDNF expression and prevented the effect of ROF on extinction recall. These effects were not associated with changes in anxiety-like behavior or general exploratory behavior. Altogether, these findings suggest that cAMP-PKA activity in the IL during the late consolidation phase after extinction learning underlies the persistence of extinction.

Cyclic-AMP Is an Actionable Novel Regulator of PD-L1 Expression in Untransformed Lymphocytes and Diffuse Large B Cell Lymphomas

Antigen-specific T lymphocytes can recognize and eliminate aberrant cells. Cancer cells halt this process by hijacking a system of immune checkpoints, the programmed cell death 1 (PD-1) and its ligands (PD-L1/2) pathway, which physiologically regulates the quantity and activity of T cells, establishing peripheral T cell tolerance and limiting tissue damage. PD-L1-expressing cancer cells interact with and inhibit PD-1 positive T cells, thus abrogating anti-cancer immunity, which can be restored by checkpoint inhibitors (CPI). Improved understanding of the regulation of PD-L1 expression will shed further light on how cancer cells escape immune surveillance, and it may help in the design of combinatorial therapeutic strategies that expand the activity of CPI. Oncogenes (e.g., MYC, STAT3, HIF1 and NF-KB) have been shown to directly induce PD-L1 transcription. In addition, pro-inflammatory cytokines, notably IFN-γ, via the JAK/STAT pathway, also increase PD-L1 expression, an intuitive counteracting regulatory axis that prevents unchecked inflammation and auto-immunity. The second messenger cyclic-AMP (cAMP) is a classical mediator of anti-inflammatory and immunosuppressive inputs. However, its putative role in PD-L1 regulation is unknown. Addressing this knowledge gap is especially relevant because this signaling node can be modulated with a class of FDA-approved agents, the phosphodiesterase 4 (PDE4) inhibitors. We have recently reviewed the pleiotropic roles that cAMP/PDE4 plays in diffuse large B-cell lymphoma (DLBCL) biology (BloodPMID: 27756749). Thus, to examine if cAMP modulates PD-L1 expression, we first used DLBCL cell lines (n=10). Raising the levels of intracellular cAMP readily induced PD-L1 expression (measured by WB and FACS) in ABC-DLBCLs but not in GCB-DLBCLs. This cAMP-mediated induction of PD-L1 occurred also at RNA level; however, using reporter assays we found that the canonical cAMP-PKA-CREB pathway does not directly activate the PD-L1 promoter. The immune modulatory activity of cAMP is mediated, at least in part, by transcriptional activation/secretion of cytokines. Thus, we considered that cAMP induction of PD-L1 in DLBCL may be driven by an autocrine loop. In agreement with this idea, cAMP promoted JAK/STAT activation and culturing DLBCL cell lines in conditioned media (CM) from cAMP-high models induced PD-L1 expression. These assays pointed to secreted factor(s) as intermediaries in the cAMP/PD-L1 axis. Therefore, we screened a panel of 105 cytokines to identify those secreted by DLBCL cell lines following cAMP up-modulation - in most models, we detected a significant cAMP-driven increase in IL-6, IL-8, IL-10 and IL-1α secretion. For validation, we focused on IL-10 because this was the most commonly cAMP-induced cytokine across the DLBCL models. We found that recombinant IL-10 induced PD-L1, albeit this induction was significantly less marked than that observed following an increase in intra-cellular cAMP. Concordantly, antibody-based blocking of the IL-10 signals, and pharmacologically inhibiting the JAK/STAT pathway, only partially abrogated the cAMP-mediated induction of PD-L1. We concluded that IL-10 and JAK/STAT signals relay part, but not all, of the cAMP effects on PD-L1 expression in DLBCL. Next, we utilized the Pde4b null mouse model to examine if these observations were present in an organismal level and in non-immortalized immune cells. In these assays, spleens of Pde4b WT, +/- and -/- mice (8-16 weeks old, male and female, n=8) were collected and analyzed by WB and FACS. Spleen cells from Pde4b deficient mice had markedly higher expression of PD-L1 (WB). By FACS, we found that the increase in PDL1 expression in Pde4b null mice derived from T cells, B cells, but from the smaller non-B/T cell population (CD19/CD3 negative). Finally, we found that the PDE4 inhibitor roflumilast used as a single agent in vitro robustly induced PD-L1 expression in DLBCL cell lines. In summary, we identified cAMP as an "actionable" novel regulator of PD-L1 expression in normal and malignant immune cells. Mechanistically, cAMP drives an autocrine loop enacted by cytokines and transduced in part by JAK/STAT. This finding supports the clinical testing of roflumilast to induce PD-L1 expression, a strategy that may improve the activity of checkpoint inhibitors in DLBCL and related tumor types. Disclosures No relevant conflicts of interest to declare.

Phosphodiesterase-4 Inhibitor Roflumilast Attenuates Pulmonary Air Emboli–Induced Lung Injury

BackgroundPulmonary air embolism (PAE)–induced acute lung injury (ALI) can be caused by massive air entry into the lung circulation. PAE can occur during diving, aviation, and some iatrogenic invasive procedures. PAE-induced ALI presents with severe inflammation, hypoxia, and pulmonary hypertension, and it is a serious complication resulting in significant morbidity and mortality. Phosphodiesterase-4 (PDE4) inhibitors can regulate inflammation and are therefore expected to have a therapeutic effect on ALI. However, the effect of the PDE4 inhibitor roflumilast on PAE-induced ALI is unknown. MethodsThe PAE model was undertaken in isolated-perfused rat lungs. Four groups (n = 6 in each group) were defined as follows: control, PAE, PAE + roflumilast 2.5 mg/kg, and PAE + roflumilast 5 mg/kg. Induction of PAE-induced ALI was achieved via the infusion of 0.7 cc air through the pulmonary artery. Roflumilast was administered via perfusate. All groups were assessed for pulmonary microvascular permeability, lung histopathology changes, pulmonary edema (lung weight/body weight, lung wet/dry weight ratio), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-17, nuclear factor–kappa B (NF-κB), and inhibitor of NF-κB alpha (IκB-α). ResultsAfter the induction of air, PAE-induced ALI presented with pulmonary edema, pulmonary microvascular hyperpermeability, and lung inflammation with neutrophilic sequestration. The PAE-induced ALI also presented with increased expressions of IL-1β, IL-6, IL-8, IL-17, TNF-α, and NF-κB and decreased expression of IκB-α. The administration of roflumilast decreased pulmonary edema, inflammation, cytokines, NF-κB, and restored IκB-α level. ConclusionsPAE-induced ALI presents with lung inflammation with neutrophilic sequestration, pulmonary edema, hyperpermeability, increased cytokine levels, and activation of the NF-κB pathway. Roflumilast attenuates lung edema and inflammation and downregulates the NF-κB pathway and cytokines.

Inhibition of phosphodiesterase‐4D reverses memory deficits and depression‐like effects via cAMP signaling in mouse models of Alzheimer's disease

Depression is highly correlated with Alzheimer's disease (AD), but treatment for the comorbidity of both is lacking. Phosphodiesterase‐4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP (cAMP), has been considered as a promising target for treatment of both memory loss in AD and depression. Using mouse models of AD and potent PDE4 inhibitors and PDE4‐subtype knockout mice, we demonstrated that PDE4D plays a role in the comorbidity of memory loss and depression in AD. Treatment with the PDE4 inhibitor rolipram or roflumilast reversed memory deficits in novel object recognition and Morris water‐maze tests in APP/PS1 double transgenic and 3xTg‐AD mice, which are widely used models for AD. Similarly, in the tail‐suspension and forced‐swimming tests, the PDE4 inhibitors reversed the decreased immobility in AD mice, suggesting antidepressant‐like effects. The effects of PDE4 inhibitors were mimicked in the tests using mice deficient in PDE4D, which displayed memory‐enhancing and antidepressant‐like effects, relative to the WT mice microinfused with beta‐amyloid peptide 1–42 into the hippocampus. In addition, the PDE4 inhibitor treatment reversed the decreased ratio of Bcl‐2/BAX and inhibited the increased expression of PDE4D in the cerebral cortex and hippocampus of AD mice. Further, the PDE4 inhibitors reversed the decreased levels of cAMP and expression of phosphorylated cAMP response element‐binding protein (CREB) and brain derived neurotrophic factor (BDNF) in AD mice. These were mimicked by PDE4D deficiency and treatment with a PDE4D relatively selective inhibitor. In conclusions, these results suggest that PDE4 is an important target for the comorbidity of memory loss and depression in AD, which appears to be mediated by PDE4D‐cAMP signaling.Support or Funding InformationThis work was supported by research grants from NIH/NIA AG031687 and NIH/NIAAA HHSN275201700001C (both to HTZ) and the National Natural Science Foundation of China (81441111 to HW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute treatment with the PDE4 inhibitor roflumilast improves verbal word memory in healthy old individuals: a double-blind placebo-controlled study

There is ample evidence that phosphodiesterase 4 (PDE4) inhibition can improve memory performance in animal studies. In the present study, we examined the acute effects of the PDE4 inhibitor roflumilast on memory performance in healthy individuals (60–80 years of age). We tested the effects of acute roflumilast administration (100, 250, 1000 μg) in a double-blind, placebo-controlled, 4-way crossover design. Participants were first screened for their verbal word memory performance to ensure normal memory performance (within 0.5 standard deviation from norm score; n = 20) Drug effects on memory performance were tested in a verbal memory test and a spatial memory test. Reported side effects of drug treatment were registered. Roflumilast (100 μg) improved the delayed recall performance of the participants (Cohen's d, 0.69). No effects were observed in the spatial memory task. Roflumilast was well tolerated at this low dose. Although no clear adverse side effects were reported at the low dose, mild adverse events (including headache, dizziness, insomnia, and diarrhea) were reported after the 1000 μg dose. The present study provides first evidence that the PDE4 inhibitor roflumilast improves verbal memory performance in old participants. The current data encourage further development of PDE4 inhibitors for improving memory.

Phosphodiesterase 4D inhibition boosts remyelination in multiple sclerosis

Event Abstract Back to Event Phosphodiesterase 4D inhibition boosts remyelination in multiple sclerosis Melissa Schepers1, 2, Dean Paes1, 2, Assia Tiane1, 2, Evelien Houben1, Olga Bruno3, Chiara Brullo3, Niels Hellings1, Jos Prickaerts2 and Tim Vanmierlo1, 2* 1 University of Hasselt, Belgium 2 School for Mental Health and Neuroscience, Maastricht University, Netherlands 3 Sezione di Chimica del Farmaco, Dipartimento di Farmacia, University of Genova, Italy Progressive multiple sclerosis (pMS) is a chronic demyelinating disorder of the central nervous system (CNS). pMS is featured by failing remyelination processes due to the inability of oligodendrocyte precursor cells (OPC) to differentiate into myelin-forming oligodendrocytes. Phosphodiesterase 4 (PDE4) is a family of enzymes that hydrolyze and inactivate cyclic adenosine monophosphate (cAMP), a second messenger crucially involved in OPC differentiation. We recently found that the aspecific PDE4 inhibitor roflumilast induces in vitro and in vivo remyelination. Yet, the use of aspecific PDE4 inhibitors coincides with emetic side-effects at the repair-inducing dose. Therefore, we hypothesize that selective inhibition of PDE4D promotes remyelination at non-emetic doses. The PDE4D inhibitor Gebr32a (5µM) accelerated in vitro OPC differentiation and showed accelerated ex vivo remyelination in lysolecithin-demyelinated mouse brain slices. Next, 10-week-old male C57bl6 mice were subjected to demyelination in the cuprizone model (6 weeks, 0.3% cuprizone w/w). Upon withdrawal of the cuprizone, mice were treated with either with Gebr32a (7days: 0.1mg/kg or 0.3mg/kg) or vehicle. Compared to vehicle, Gebr32a 0.3mg/kg treatment displayed an enhanced remyelination by yielding an increased expression of myelin basic protein (MBP) in the corpus callosum and dendate gyrus while improving memory performance in the object location task (OLT) . The improved remyelination and spatial memory performance following Gebr32a treatment at a non-emetic dose, prompt us to conclude that PDE4D inhibition boosts the endogenous repair mechanisms in cuprizone fed mice and thereby improves cognitive performances. Keywords: Phosphodiesterase (PDE), Multiple scleorsis (MS), Cognition, oligodendrocyte precursor cell (OPC), CNS repair Conference: 13th National Congress of the Belgian Society for Neuroscience , Brussels, Belgium, 24 May - 24 May, 2019. Presentation Type: Poster presentation Topic: Cellular/Molecular Neuroscience Citation: Schepers M, Paes D, Tiane A, Houben E, Bruno O, Brullo C, Hellings N, Prickaerts J and Vanmierlo T (2019). Phosphodiesterase 4D inhibition boosts remyelination in multiple sclerosis. Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience . doi: 10.3389/conf.fnins.2019.96.00034 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Mx. Tim Vanmierlo, University of Hasselt, Hasselt, Belgium, tim.vanmierlo@uhasselt.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Melissa Schepers Dean Paes Assia Tiane Evelien Houben Olga Bruno Chiara Brullo Niels Hellings Jos Prickaerts Tim Vanmierlo Google Melissa Schepers Dean Paes Assia Tiane Evelien Houben Olga Bruno Chiara Brullo Niels Hellings Jos Prickaerts Tim Vanmierlo Google Scholar Melissa Schepers Dean Paes Assia Tiane Evelien Houben Olga Bruno Chiara Brullo Niels Hellings Jos Prickaerts Tim Vanmierlo PubMed Melissa Schepers Dean Paes Assia Tiane Evelien Houben Olga Bruno Chiara Brullo Niels Hellings Jos Prickaerts Tim Vanmierlo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The phosphodiesterase-4 inhibitor roflumilast reverts proteolysis in skeletal muscle cells of patients with COPD cachexia.

Peripheral muscle weakness and mass loss are characteristic features in severe chronic obstructive pulmonary disease (COPD). We hypothesized that the phosphodiesterase (PDE)-4 inhibitor roflumilast-induced cAMP may ameliorate proteolysis and metabolism in skeletal muscles of COPD patients with severe muscle wasting. In myogenic precursor cells (isolated from muscle biopsies and cultured up to obtain differentiated myotubes) from 10 severe COPD patients and 10 healthy controls, which were treated with 1 μM roflumilast N-oxide (RNO) for three time cohorts (1, 6, and 24 h), genes of antioxidant defense and oxidative stress marker, myogenesis and muscle metabolism, proteolysis (tyrosine release assay) and ubiquitin-proteasome system markers, autophagy, and myosin isoforms were analyzed using RT-PCR and immunoblotting. In COPD patients at 6 h RNO treatment, myotube tyrosine release, total protein ubiquitination, and tripartite motif-containing protein 32 levels were significantly lower than healthy controls, whereas at 24 h RNO treatment, myotube myosin heavy chain ( MyHC) -I and MyHC-IIx expression levels were upregulated in both patients and controls. In the 6-h RNO cohort, in patients and controls, myotube expression of nuclear factor (erythroid-derived 2)-like 2 ( NRF2) and its downstream antioxidants sirtuin-1, FGF-inducible 14, and insulin-like growth factor-1 was upregulated, whereas that of myocyte-specific enhancer factor 2C, myogenic differentiation, myogenin, myostatin, atrogin-1, and muscle RING-finger protein-1 was downregulated. In myotubes of severe COPD patients with cachexia, roflumilast-induced cAMP signaling exerts beneficial effects by targeting muscle protein breakdown (tyrosine release), along with reduced expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In both patients and controls, roflumilast also favored antioxidant defense through upregulation of the NRF2 pathway and that of the histone deacetylase sirtuin-1, whereas it improved the expression of slow- and fast-twitch myosin isoforms. These findings show that muscle dysfunction and wasting may be targeted by roflumilast-induced cAMP signaling in COPD. These results have potential therapeutic implications, as this PDE-4 inhibitor is currently available for the treatment of systemic inflammation and exacerbations in patients with severe COPD. NEW & NOTEWORTHY In myotubes of cachectic chronic obstructive pulmonary disease (COPD) patients, cAMP signaling exerted beneficial effects by targeting muscle proteolysis and reducing gene expression of proteolytic markers of the ubiquitin-proteasome system and that of myostatin. In myotubes of patients and controls, roflumilast also favored antioxidant defense through upregulation of the nuclear factor (erythroid-derived 2)-like 2 pathway, of sirtuin-1, and of gene expression of slow- and fast-twitch isoforms. These findings have potential clinical implications for the treatment of muscle wasting in patients with COPD and cachexia.

The PDE4 inhibitor roflumilast reduces weight gain by increasing energy expenditure and leads to improved glucose metabolism.

To investigate the metabolic effects of the phosphodiesterase-4 (PDE4) inhibitor roflumilast, a clinically approved anti-inflammatory drug used for the treatment of chronic obstructive pulmonary disease. The metabolic effects of roflumilast were investigated in C57BL/6J mice, fed a high-fat Western-type diet and treated with or without roflumilast for a period of 12 weeks. Roflumilast led to a marked reduction in body weight gain, which became apparent in the second week after treatment initiation and was attributable to a pronounced increase in energy expenditure. Furthermore, roflumilast improved glucose tolerance, reduced insulin resistance and diminished steatohepatitis in mice. Mechanistically, this was associated with hepatic protein kinase A (PKA) and cAMP response element binding protein (CREB) activation, leading to peroxisome proliferator-activated receptor gamma coactivator-1α (PCG-1α)-dependent induction of mitochondrial biogenesis. Consistently, roflumilast increased the cellular respiratory capacity of hepatocytes in a PKA-dependent manner. Roflumilast-dependent PDE4 inhibition is a new target for weight loss strategies, especially in conditions of associated comorbidities such as insulin resistance and non-alcoholic steatohepatitis.