Phosphodiesterase 4 in inflammatory diseases: effects of apremilast in psoriatic blood and in dermal myofibroblasts through PDE4/CD271 complex

Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, while PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared to normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis, as compared to normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by β-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF-and TGF b 1 -induced fibroblasts migration, and inhibits DF differentiation into DM mediated by NGF or TGF b 1 . Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with β-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the complex PDE4/CD271 could be important in modulating fibroblasts functions.

However, the expression patterns of PDE4 isoforms have not been studied in blood or skin of patients with inflammatory disease such as psoriasis.
PDE4 inhibitors are a well-characterized class of pharmaceutical agents, with a broad range of anti-inflammatory activities in vitro, and in in vivo preclinical models of asthma, lung neutrophilia, arthritis, inflammatory bowel disease, multiple sclerosis, osteoporosis and other conditions (Videla S,.Chem. 2009;47:37-74. Review. PubMed PMID:19328289).Apremilast is a novel, orally administered, small molecule phosphodiesterase 4 (PDE4) inhibitor approved for treatment of psoriatic arthritis (PsA) and moderate to severe plaque psoriasis CD271 is a neurotrophin (NT) receptor, also known as p75 NTR , which belongs to the TNF receptors superfamily and shares with the other members of the family the death domain.The role of CD271 is complex: it may increase high-affinity binding of Trk receptors to NTs, and enhances their survival signal.By contrast, CD271 alone can induce apoptosis upon specific binding, such as with β-amyloid, via its own transduction pathway and by interacting with a mounting number of downstream molecules (Dechant G. Nat Neurosci. 2002Nov;5(11):1131-6. Review. PubMed PMID: 12404007).NT and their receptors have autocrine and paracrine functions within the skin (Botchkarev VA.J Invest Dermatol. 2006 Aug;126(8):1719-27. Review. PubMed PMID: 16845411).Moreover, our group has recently demonstrated that CD271 is expressed by basal keratinocytes, specially by transit amplifying (TA) cells (Truzzi et al, 2011, 18:948-958 Cell Death and Diff.PMID: 21151024).It should be noted that the crosstalk between the epidermal and dermal compartment is necessary to sustain epidermal homeostasis, and that some factors that are produced in the dermal compartment act on stem cells that reside in the epidermis.Moreover, Palazzo et al showed that both dermal human fibroblasts and myofibroblasts express NT, CD271 and Trk receptors and that NTs exert a critical role in tissue remodelling and wound healing (Palazzo E et al 2012. PubMed PMID 21503896) In the current set of experiments, we report gene expression patterns of PDE4 isoforms (A,B,C,D) in peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with a variety of immune-mediated or inflammatory diseases including psoriasis, as well as the effect of apremilast on cytokine and chemokine production from PBMC of patients with psoriasis.We further report immunohistochemistry studies showing the isoform distribution of PDE4 in skin samples from healthy donors and psoriasis, atopic dermatitis and discoid lupus patients.We then examine PDE4 isoform binding to CD271 in fibroblasts cultured from healthy skin, and analyze the function of the CD271-PDE4 complex in fibroblasts.

Gene Expression Analysis of PDE4 isoforms in PBMC
Viably frozen PBMC and autologous serum from healthy volunteers or patients with psoriasis, RA, IPF, sarcoidosis, scleroderma, Crohn's disease, or SLE (n=5 from each disease) were purchased from Coversant Healthcare Systems (Huntsville, Alabama).The diagnosis of disease was made on a case report form by the responsible physician in a network of medical clinics, research clinics, hospitals, and outpatient medical facilities.PBMC were thawed and cultured in RPMI-1640 supplemented with 5% of each patient's autologous serum for 24 hours to allow cells to recover from freezing and thawing.Cells (1x10 6 PBMC per patient) were collected for gene expression analysis by hybridization array.Cells were washed with cold phosphatebuffered saline, lysed with 350 μL RLT buffer (Qiagen, Gaithersburg, Maryland), and transferred to barcoded tubes.RNA was extracted and converted to cDNA using the Ovation Whole Blood Solution (NuGEN Technologies, San Carlos, California), and hybridized to HG-U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, California).Gene expression data were normalized and provided by Covance Genomics Laboratory (Seattle, Washington).Statistical comparison for PBMC from each disease versus normal control was by one-way ANOVA.

qRT-PCR
Quantitative RT-PCR was performed to confirm overexpression of PDE4B and PDE4D isoforms in PBMC from patients with psoriasis.PBMC samples (n=20 each from psoriasis patients and healthy donors) were cultured for 24 hours in 5% autologous serum.RNA was isolated, converted to cDNA and analyzed by qRT-PCR, and a comparison of PBMC from psoriasis patients versus normal controls was conducted by t-test.

Multiplexed Bead-Based Immunoassay
PBMCs from psoriasis patients (n=10) were obtained after informed consent by Conversant Healthcare Systems (Huntsville, Alabama).The diagnosis of psoriasis was made on a case report form by the responsible physician in a network of medical clinics, research clinics, hospitals, and outpatient medical facilities.PBMCs were thawed (minimum viability was 80%), plated at 2x10 5 cells/well, and stimulated in duplicate via various immune receptors with or without apremilast (1 μM) in 0.1% dimethyl sulfoxide for 24 hours.Immune cell stimuli included 1 ng/mL LPS (Sigma), targeting TLR4 on monocytes, dendritic cells, and B cells; 10 ng/mL each of staphylococcal enterotoxin A and B (Toxin Tech), targeting T-cell receptors; 2 ng/mL each of TNF-α + IL-1β (Sigma), targeting TNF and IL-1 receptors on monocytes, dendritic cells, B cells, T cells, and natural killer cells; and 2 μg/mL anti-IgM (Jackson IR), targeting B-cell receptors.
The production of 25 protein analytes was measured by cytometric bead array using Milliplex MAP Human Cytokine/Chemokine beads (EMD Millipore Corporation, Billerica, MA) on a MagPix instrument (Luminex, Austin, TX).

Skin Immunohistochemistry (IHC)
Immunohistochemistry was performed on dermis samples from healthy controls and from patients with psoriasis, atopic dermatitis (AD), and discoid lupus erythematosus (DLE) at University of Modena and Reggio Emilia, Modena, Italy.

Isolation of primary fibroblasts and cell treatment
Normal human dermal fibroblasts (DF) were isolated from healthy skin biopsies obtained from waste materials from Operating Room.Patient consent for experiments was not required because Italian laws considers human tissue left over from surgery as discarded material.Briefly, DF were obtained by explant culture and grown in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS).

Immunofluorescence (IF) in situ
DF were plated on chamber slides and, 48 hours after seeding, were treated with TGFβ 1 (1 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) or diluent alone.Six days later, cells were fixed in situ in 4% paraformaldehyde for 20 min and air dried.After a rehydration in PBS, cells were permeabilized for 10 min with 0.5% Tryton X-100 in 0.1% sodium citrate, treated for 5 min with 50 mM NH4Cl and incubated with 1% bovine albumin serum for 20 min.Then slides were incubated for 1 hour at room temperature with the mouse monoclonal anti-α-SMA antibody (1:400, Sigma-Aldrich).Then cells were incubated with secondary antibody, Alexa Fluor 488 (1:100, Thermo Fisher Waltham, MA, USA).Finally, cell nuclei were stained with 1μg/ml Dapi (Sigma-Aldrich).

Co-Immunoprecipitation
DF and DM were cultured until pre-confluent condition.Cells were harvested with lysis buffer pH 7.5 (150 nM NaCl, 15 mM MgCl, 1 mM EGTA, 50 mM Hepes, 10% glycerol, 1% Triton) containing protease inhibitors.Monoclonal antibody CD271 (Upstate, Lake Placid, New York), was bound to sepharose beads for 1 h at 4 ºC under rotation.Thereafter, pre-cleared lysates were conjugated to sepharose beads-antibody complex or, as control, to sepharose beads alone or to antibody alone, overnight at 4 ºC under rotation.Immunocomplexes were washed with binding buffer (50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 2 mM MgCl 2 , 1 mM DTT, 0.5% Triton X-100, 0.5% bovine serum albumin) six times.Samples were eluted with 2x Laemnli sample buffer for 5 minutes at 90 ºC and Western blotting for PDE4A, B, C and D was performed as described below.

MTT Assay
DF and DM were plated in a 96-well tissue culture plate (5000 cells/well).48 hours after plating, cells were treated as described above with apremilast or DMSO for 2 hours, then treated with diluent (control), NGF (100 ng/mL), NT3 (100 ng/mL), or β-amyloid (40 μM).MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed 48 hours later.Proliferative cells were detected by incubating with MTT (Sigma-Aldrich) solution at 37 ºC for 4 hours.The formazan dye produced after DMSO solubilization was evaluated by a multiwall scanning spectrophotometer at 540 nm.The results are expressed as viability percentage, as compared to control.Results are calculated as the mean ± SD of three different experiments.Student's t-test was performed for comparison of the means.
After 15 minutes, cells were analyzed using an Epics XL flow cytometer (Coulter Electornics Inc., Hialeah, FL).Apoptosis was detected by evaluating the fluorescence of hypodiploid cells counted in subG1 region.

TUNEL Assay
DF and DM after treatment were fixed in situ in paraformaldehyde (4% in PBS) and airdried.The slides were stained with the "In situ cell death detection kit" (Roche Diagnostics, Basel, Switzerland) as recommended by the manufacturer.Fluorescent specimens were analyzed by confocal scanning laser microscopy (Leica TCS SP2, Leica microsystems, Wetzlar, Germany) in conjunction with a conventional optical microscope (Leica DM IRBE, Leica).Results are calculated as the mean ± SD of three different experiments.

Migration Assay
A total of 20x10 4 DF were plated on 12-well tissue culture plates.48 hours after seeding, cells were pretreated with DMSO or apremilast for 2 hours as described above, then cells were treated with 5 mg/mL mitomycin C for 1 hr 30 min.Subsequently, the cells were washed three times in serum-free medium and a line for each well was drawn along the cell monolayer with a sterile plastic tip.Plates were washed twice with serum-free medium to remove all detached cells and incubated in medium with BSA 0.1% with 1 ng/mL TGF-β 1 , 100 ng/mL human recombinant NGF in presence of DMSO or apremilast.Cells were monitored at 24 and 48 hours from stimulation.The result of each experiment was expressed as the mean of migrated cells from three different areas.The final results are expressed as the mean ± SD of three different experiments.

cAMP ELISA
At 48 hours after cell seeding, DF and DM were pretreated with DMSO or apremilast as described above, then diluent, 100 ng/mL human recombinant NGF (Sigma-Aldrich), or 40 μM β-amyloid (Bachem) were added to the culture medium.48 hours later, supernatants were collected, particulates were removed by centrifugation and stored at -20 ºC.cAMP quantitation was performed by a competitive enzyme immunoassay (R&D Systems cAMP immunoassay, R&D Systems, Minneapolis, MN) according to manufacturer instructions.The sample concentration was determined by absorbance at 540 nm against a standard curve.cAMP levels are given in pmol/mL of cell lysate and results are expressed as mean ± SEM of triplicate from three different experiments.

Gene expression analysis of PDE4 isoforms in PBMC
We first wanted to analyze gene expression of PDE4 isoforms (A, B, C, and D) in PBMC from healthy donors and from patients with 7 different immune-mediated or inflammatory diseases: psoriasis, rheumatoid arthritis (RA), idiopathic pulmonary fibrosis (IPF), sarcoidosis, scleroderma, Crohn's disease, and systemic lupus erythematosus (SLE).PDE4B and PDE4D isoforms were increased at the mRNA level in PBMC from psoriasis patients relative to normal controls (Figure B and D, p<0.05).
PDE4D was also increased versus normal controls in PBMC from patients with SLE (Figure 1D, p<0.05).PDE4C mRNA was preferentially overexpressed in PBMC from patients with Crohn's disease versus normal controls (Figure 1C), while PDE4A mRNA expression levels were similar across PBMC from normal controls and patients with the various immune/inflammatory diseases (Figure 1A).To confirm the overexpression of PDE4B and PDE4D mRNA in PBMC from patients with psoriasis relative to normal controls, quantitative RT-PCR was performed using 20 samples each from psoriasis patients and healthy donors.Mean relative gene expression in PBMC from psoriasis patients was 5.4-fold higher for PDE4B, and 2.2-fold higher for PDE4D (p<0.0001 for both vs normal control), compared with 1.1-fold for PDE4A (p=NS) (Figure 1E).There were no significant correlations between PDE4A, PDE4B, or PDE4D gene expression in the psoriatic PBMC with PASI, BSA, or sPGA scores (data not shown).

Effect of apremilast on the production of inflammatory mediators by PBMC from patients with psoriasis
Given the high PDE4 expression in psoriasis, we investigated the effect of

PDE4 expression in inflammatory skin infiltrates
Psoriasis is an immune-mediated disease characterized by a dense dermal infiltrate extravasating from the blood stream.Immuno-inflammatory infiltrate in turn is responsible for the epidermal alterations.For this reason, we focused on the expression of PDE4 proteins, that are markedly elevated in PBMC from psoriatic patients, in the dermal infiltrate from lesional psoriatic skin.Also dermis from healthy subjects, atopic dermatitis (AD), and discoid lupus erythematosus (DLE) was evaluated.In general, PDE4 staining was higher in diseased vs. normal skin when measured by ImageJ software (Figure 3B).PDE4A was expressed at the cytoplasmic level in a few dermal cells, most likely fibroblasts, in healthy skin, while it was markedly increased in fibroblasts from psoriasis, AD, and discoid lupus erythematous (DLE).PDE4B was slightly expressed by dermal cells both in healthy and psoriatic skin.In AD, PDE4B staining was somewhat more expressed in the dermis of AD skin as compared to controls.In this context, dermal fibroblasts appeared intensely positive.In dermis from DLE skin, PDE4B staining was markedly increased, as compared to healthy controls, dermal fibroblasts being strongly positive.PDE4C was considerably more expressed in the dermis from psoriatic or AD sections than in skin from healthy controls.In DLE, PDE4C staining intensity was higher than in AD or psoriasis, fibroblasts being intensely stained.PDE4D expression was markedly higher in dermal infiltrate (fibroblasts) from psoriasis, AD or DLE than in healthy skin.Finally, CD271 appeared more expressed in fibroblasts from psoriasis and AD than in healthy skin or DLE (Figure 3A).
In order to better characterize PDE4 protein expression in psoriatic skin, a more detailed analysis was performed in the dermis.PDE4A, B, and D staining was evaluated and interpreted by a pathologist, and each antibody was evaluated for the presence of specific signal and level of background.Staining intensity was recorded on a 0-4 scale (0=negative, 1=blush, 2=faint, 3=moderate, 4=strong) (Table 1).Overall, lymphocytes present in the psoriatic skin were moderately to strongly positive for PDE4A and PDE4B, and faintly positive for PDE4D.Pilosebaceous units, sweat glands, sweat ducts, hair follicles, and arrector pili smooth muscle were positive.Fibroblasts were positive, and endothelium stained strongly for PDE4A, PDE4B, and PDE4D.Specifically, PDE4A expression showed increased intensity and prevalence of staining in nearly all cell types in psoriatic vs. normal samples, although neutrophils within microabscesses were exceptionally negative.PDE4B staining in psoriatic samples was very prominent in vessels in the superficial dermis, and positively stained inflammatory cells were more abundant than in normal samples.PDE4D staining in psoriatic samples was present in fibroblasts and endothelium.Lymphocytes showed frequent staining in both normal and psoriatic skin samples, but were far more prevalent in the psoriasis samples.The endothelium of psoriatic skin showed a relatively large increase in PDE4 expression compared to normal skin, with PDE4A and PDE4D displaying strong staining in psoriatic endothelium, but only faint staining in normal endothelium.In fibroblasts, PDE4A expression was moderate to strong in psoriatic skin, but absent in normal skin.PDE4D expression was moderate in psoriatic fibroblasts, but faint in normal fibroblasts.In overall terms of staining intensity, the endothelium and fibroblasts showed the largest consistent increase in PDE4 expression in psoriatic vs. normal skin.

PDE4 isoforms co-immunoprecipitate with CD271 in fibroblasts
Due to the large increase in PDE4 staining observed in psoriatic fibroblasts, this cell type was selected for functional studies to determine the role of PDE4 in cellular responses.Different proinflammatory cytokines, including TGF-b 1 , promote fibroblast differentiation into myofibroblasts, characterized by the expression of a-Smooth Muscle Actin (a-SMA).These cells are predominantly responsible for connective-tissue contracture and ECM reorganization in different physiopathologic processes in the skin, including wound healing and fibrosis (Verjee et al 2013/ PMID:23431165).We showed that all PDE4 isoforms are expressed in dermal fibroblasts (DF) and myofibroblasts (DM) cultured from healthy donors at the mRNA level (Figure 4A, B, C), while PDE4 protein levels are higher in DM than in DF (Figure 4D).PDE4A had previously been found to associate with the CD271 to enhance cAMP degradation in fibroblasts, and to regulate fibrotic responses [Sachs BD, Baillie GS, McCall JR, et al. J Cell Biol. 2007 Jun 18;177(6):1119-32. PubMed PMID: 17576803].Because CD271 is also expressed in skin fibroblasts (Palazzo et al, 2012, J Cell Physiol), we wanted to determine if PDE4 isoforms (PDE4A,B,C, and D) co-localize with CD271 in dermal fibroblasts.We showed that PDE4 isoforms co-immunoprecipitate with CD271 in DM (Figure 4E).However, in DF, CD271 formed only weak complexes with PDE4A and PDE4B and no complex with PDE4C or PDE4D.

PDE4-CD271 complex function in fibroblasts in vitro
To evaluate the effect of the PDE4-CD271 complex on proliferation and apoptosis in fibroblasts, DF and DM were pretreated with apremilast or DMSO for 2 hours and then treated with NGF, NT3, β-amyloid, or diluent.Treatment with β-amyloid significantly decreased the percentage of proliferative cells for both DF and DM cultures compared with diluent control.In DM, the addition of apremilast significantly inhibited the effect of β-amyloid (p<0.05vs DMSO), while apremilast had no effect versus DMSO in DF (Figure 5A, B).Similarly, the addition of apremilast significantly reduced the induction of apoptosis by β-amyloid treatment in DM (p<0.05 vs DMSO, Figures 5D, F) but not in DF (Figures 5C E).
It has been shown previously that neurotrophins mediate DF migration, and that neurotrophins as well as TGF-β 1 induce DF to differentiate into DM by expressing α-SMA.{Palazzo,2012 3572 /id} We therefore analyzed whether the addition of apremilast could affect DF migration and α-SMA expression.Apremilast inhibited DF migration induced by both NGF and TGF-β 1 treatment (Figure 6A, B).Similarly, apremilast inhibited the up-regulation of α-SMA expression induced by NGF and TGF-β 1 , especially 6 days after treatment (Figure 6C).cAMP ELISA was performed to evaluate if activation of the CD271-PDE4 complex leads to cAMP degradation.In this analysis, neither apremilast alone nor apremilast in combination with NGF exerted any significant effect on release of cAMP by either DF or DM (Figure 7A).On the other hand, in DM, Apremilast reduced cAMP degradation induced by treatment with β-amyloid (Figure 7B Because of the higher PDE4 gene expression observed in psoriasis patient blood, a careful analysis of the skin was conducted.PDE4 protein expression is higher in the skin of patients psoriasis, atopic dermatitis, and discoid lupus, as compared to normal skin.In a detailed IHC analysis of PDE4 protein expression in psoriatic skin, the endothelium and fibroblasts, in particular, shows increased expression of PDE4A and PDE4D, as compared to normal skin.Little had previously been known about the expression of PDE4 isoforms in human dermal fibroblasts.Selige and co---workers showed that primary normal human lung fibroblasts (NHLF) express PDE4A, B, and D, while PDE4C is only barely detected (Selige J, J Cell Physiol.2011Physiol. , 226:1970-80.)-80.).In the present study, we found that all PDE4 isoforms are expressed in both DF and DM, even if at different levels.Regarding dermal compartment, all PDE4 isoforms are expressed at the mRNA level both in DF and DM.DM express higher level of PDE4 isoform proteins than DF.DM invade and repair injured tissues Myofibroblasts.I. Paracrine cells important in health and disease.Am J Physiol.1999, 277:C1---9.).We found that all four PDE4 proteins are up-regulated in cells of the dermal compartment in biopsies from inflammatory conditions.We postulate that these cells could be DM, and suggest that PDE4 proteins play a role in the above mentioned DM functions, although more functional studies are definitely needed.
Because PDE4 isoforms are expressed in dermal fibroblasts and evidence has indicated an interaction between PDE4 and the CD271, which may be important in tissue remodeling and wound healing,{Palazzo, 2012 3572 /id}, we assessed the effect of PDE4 inhibition on the function of the PDE4-CD271 complex in DF and DM.We first showed all PDE4 isoforms co-immunoprecipitate with CD271 in DM.Inhibition of PDE4 with apremilast reduces cAMP degradation mediated by β-amyloid in DM, as well as βamyloid-mediated induction of apoptosis and reduction of proliferative cells .Addition of apremilast also reduces DF migration and differentiation mediated by NGF and TGF-β 1 .
Taken together, these results indicate that PDE4 play an important role in psoriasis.In addition, the study reveals that the complex PDE4/CD271 could be important in modulating fibroblasts functions.
apremilast on cytokines produced by PBMC from psoriatic patients.Psoriasis patient PBMC donors (n=10) included 5 females and 5 males ranging in age from 26 to 42 years, with PASI scores ≤26 and ≤33% body surface area affected.Two patients also had a diagnosis of psoriatic arthritis.Changes in production of various cytokines and chemokines upon stimulation of PBMCs from these psoriasis patients with LPS, staphylococcal enterotoxin A and B, TNF-α/IL-1β, or anti-IgM are shown in Figure 2A.Addition of 1 μM apremilast had the greatest effect on psoriatic PBMC cultures stimulated via TLR4 (ie, LPS stimulation), with 13 of the 25 analytes significantly affected versus LPS control (Figure 2B).Specifically, apremilast caused significant inhibition of LPS-induced TNF-α, IL-12/IL-23p40, IFN-γ, IP-10, MCP-1, MCP-3, MIP-1β, IL-1α, IL-1β, and GM-CSF production, and increased IL-10, G-CSF, and VEGF production.In psoriatic PBMC stimulated with anti-IgM to ligate the B cell receptor, apremilast decreased production of the cytokines TNF-α, IL-1α, and IFN-γ, and the chemokines MCP-1, MCP-3, MIP-1β, and IP-10, and increased IL-10, G-CSF, VEGF, and IL-6.In psoriatic PBMC cultures stimulated via the T-cell receptor or TNF and IL-1 receptor, apremilast had far fewer effects (data not shown).
In this series of experiments characterizing the role of PDE4 isoforms in psoriasis, gene expression studies indicate that PDE4B and PDE4D mRNA are overexpressed in PBMC from donor patients with psoriasis relative to normal controls (healthy volunteers) or patients with other inflammatory and immune-mediated diseases.PCR experiments confirmed that, on average, the overexpression of the PDE4B gene is 5.4-fold, and the overexpression of the PDE4D gene is 2.2-fold, compared with normal PBMC.Regarding the effect of the PDE4 inhibitor apremilast on cytokine/chemokine production of stimulated PBMCs from patients with psoriasis, apremilast has the greatest effect on responses to TLR4 receptor stimulation, with a similar pattern observed in B cell receptor-stimulated PBMC.Apremilast consistently decreases production of the cytokines TNF-α, IL-1α, and IFN-γ, and the chemokines MCP-1, MCP-3, MIP-1β, and IP-10.In psoriatic PBMC cultures stimulated via the T-cell receptor or TNF and IL-1 receptors, apremilast has far fewer effects.On the other hand, apremilast induces an up-regulation of IL-10 and G-CSF production under all conditions.
by secreting and organizing the extracellular matrix and by developing contractile forces.At the end of the normal repair process, DM disappear by apoptosis.In pathologic conditions, DM likely remain, leading to excessive scarring (Micallef L, Vedrenne N, Billet F, Coulomb B, Darby IA, Desmoulière A. The myofibroblast, multiple origins for major roles in normal and pathological tissue repair.Fibrogenesis Tissue Repair.2012, 5 Suppl 1:S5.).Indeed, DM play an important role in organogenesis, oncogenesis, inflammation, repair, and fibrosis in most organs and tissues (Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB.

FIGURE LEGENDS Figure 1 .
FIGURE LEGENDS

Figure 2 .
Figure 2. Modulation of production of inflammatory cytokines and chemokines in

Figure 5 .
Figure 5. Apremilast modulation of apoptosis in DF or DM.Fibroblast (DF) or

Figure 6 .
Figure 6.Effect of apremilast on DF migration and differentiation.DF were pretreated

Figure 7 .
Figure 7. cAMP degradation by PDE4/CD271 complex activation in DF and DM.DF