CD14++ CD16− monocytes are the main source of 11β-HSD type 1 after IL-4 stimulation

International Immunopharmacology 43 (2017) 156–163

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CD14++ CD16− monocytes are the main source of 11β-HSD type 1 after IL-4 stimulation Vidya Kunnathully a,⁎, Macarena Gomez-Lira b, Giulio Bassi c, Fabio Poli a, Elisa Zoratti d, Valentina La Verde a, Luca Idolazzi a, Davide Gatti a, Ombretta Viapiana a, Silvano Adami a, Maurizio Rossini a a

Department of Medicine, Section of Rheumatology, University of Verona, VR 37134, Italy Department of Neurological, Biomedical and Movement Sciences, Section of Biology and Genetics, University of Verona, VR 37134, Italy Department of Medicine, Section of Hematology, University of Verona, VR 37134, Italy d Applied Research on Cancer Network, University of Verona, VR 37134, Italy b c

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Article history: Received 16 October 2016 Received in revised form 23 November 2016 Accepted 10 December 2016 Available online xxxx Keywords: Monocytes IL-4 11β-HSD1 Glucocorticoids Anti-inflammatory M2

a b s t r a c t The anti-inflammatory actions of IL-4 are well established through earlier findings. However, the exact mechanism it uses to downregulate the pro-inflammatory cytokine production through monocytes and macrophages is poorly understood. In this study, we examined the effect of IL-4 in the induction of 11β-HSD1 in the two main classes of monocytes, CD14++ CD16− (CD14) and CD14+ CD16+ (CD16). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 17 healthy donors and were sorted into CD14 and CD16 subpopulations using cell sorting. Effect of IL-4 on 11β-HSD1-enzyme activity was measured in sorted and unsorted monocytes using Homogeneous Time-Resolved Fluorescence (HTRF) and M1/M2 polarization analysis was performed by flow cytometry. Our results indicate that CD14 cells are the major source of 11β-HSD1 enzyme after IL-4 stimulation and that M2 phenotype is not a pre-requisite for its synthesis. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The peripheral interconversion of pharmacologically active cortisol and inactive cortisone is accomplished by two independent 11βhydroxysteroid dehydrogenases (11β- HSDs) that exhibit tissue-specific expression [1]. 11β-HSD1 generates active cortisol from inactive cortisone and thereby enhances the activation of the glucocorticoid receptor while 11β-HSD2 is a powerful glucocorticoid inactivator. The two 11β-HSDs also interconvert inactive prednisone with active prednisolone, commonly used for the treatment of inflammatory diseases. 11β-HSD1 is broadly distributed among tissues, with predominant expression occurring in hepatic, adipose, gonadal, and central nervous system tissues. The peripheral control of inflammation involves also Abbreviations: CD14, CD14++ CD16−; CD16, CD14+ CD16+; HTRF, Homogeneous Time-Resolved Fluorescence; 11β-HSDs, 11β-hydroxysteroid dehydrogenases; CFSE, carboxyfluorescein-diacetate-succimidyl-ester method; rMFI, median fluorescence intensity. ⁎ Corresponding author at: Policlinico G.B. Rossi c/o LURM – ala ovest – lab. Reumatologia Piazzale, L.A.Scuro, 10, 37134 Verona, Italy. E-mail addresses: [email protected] (V. Kunnathully), [email protected] (M. Gomez-Lira), [email protected] (G. Bassi), [email protected] (F. Poli), [email protected] (E. Zoratti), [email protected] (V. La Verde), [email protected] (L. Idolazzi), [email protected] (D. Gatti), [email protected] (O. Viapiana), [email protected] (S. Adami), [email protected] (M. Rossini).

http://dx.doi.org/10.1016/j.intimp.2016.12.015 1567-5769/© 2016 Elsevier B.V. All rights reserved.

the conversion of inactive cortisone to cortisol (or of inactive prednisone to active prednisolone) by 11β-hydroxysteroid dehydrogenases expressed also in some immune cells [2] including dendritic cells and macrophages [3], while its expression is negligible in human blood monocytes. However, when these latter cells are exposed to IL-4 (regardless of whether the monocytes were maintained in suspension culture in Teflon beakers or as an adherent monocyte layer in plastic tissue culture dishes) the enzyme activity is tremendously enhanced, an effect abrogated by pro-inflammatory IFN-γ [3]. It has been demonstrated that IL-4 has anti-inflammatory properties, with an ability to suppress the production of tumour necrosis factor (TNF)-α and IL-1β by lipopolysaccharide (LPS)-activated human monocytes [4]. IL-4 has been successfully used for the treatment of inflammatory disease in animal models [5,6]. Recent studies suggest that therapies that cause an increase in IL-4 concentrations in tissues may be useful in the treatment of inflammation [7,8]. However, the exact mechanisms by which IL-4 exerts its anti-inflammatory effects are poorly understood. In human peripheral blood there are two main classes of monocytes, the classical CD14++ CD16− (CD14) and the pro-inflammatory CD14+ CD16+ (CD16) [9]. The CD16-negative classical monocytes form about 90% of the population, whereas the CD16-positive cells include only 10% of all monocytes under physiological conditions at rest [10]. These two kinds of monocytes have different cell surface markers and express

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differentially specific genes such as HLA-DR, Ig-like transcript 4 and others. The preferential induction of pro-inflammatory molecules from CD16 monocytes after induction with TLR2–4 ligands or LPS suggests that CD16 are the pro-inflammatory monocytes. In contrast, transcript for interleukin 10 (IL-10), are present in CD14 monocytes and almost absent in CD16 monocytes suggesting that CD14 monocytes are involved in anti-inflammatory processes [11]. 11β-HSD1 activity is tightly related to the inflammatory status of human macrophages. 11β-HSD1 gene expression is higher in pro-inflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Elevated 11β-HSD1 expression in polarized M2 macrophages might contribute to their function in the anti-inflammatory response and the resolution of inflammation, with potential consequences on inflammatory diseases [12]. It is already established that M2 macrophages show elevated levels of 11β-HSD1, but it is still unclear is the M2 phenotype is a pre-requisite for this phenomenon. Aim of this study was to determine if IL-4 induces HSD1 activity preferentially in one the two circulating monocytes subpopulations and if this observation is influenced by their polarization towards the M2 phenotype. 2. Materials and methods 2.1. Monocyte cultures Blood samples from 17 healthy donors provided by the blood transfusion center of Verona University Hospital were collected under a protocol approved by the local Ethics Committee (Comitato Etico per la Sperimentazione – AOUI) and have been performed in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standard. The freshly harvested buffy coats were processed immediately to obtain PBMCs. PBMCs were isolated by Ficoll-Paque (Stemcell technologies, Vancouver, Canada) density gradient separation. The isolated PBMCs were plated at a density 700,000 cells/well in 96 well culture plates (BD Biosciences, New Jersey, USA) and cultured for two hours in serum free RPMI-1640 (Lonza, Basel, Switzerland) medium supplemented with 2 mM L-glutamine (Gibco, Life technologies) and 50 μg/ml gentamycin (Gibco, Massachusetts, USA). After incubation cells were washed with PBS to remove lymphocytes and nonadherent cells. 2.2. Isolation of monocyte sub-populations Isolated PBMCs were stained for 15 min in the dark with a mix of the following monoclonal antibodies: anti-CD33 PE-C7 (Clone P67.6, BD Biosciences, New Jersey, USA), mouse anti-Human CD123 PE (Clone 9F5, BD Biosciences, New Jersey, USA), anti-CD16 PE (clone NKP15, BD Biosciences, New Jersey, USA) and anti-CD14 FITC (clone MϕP9, BD Biosciences, New Jersey, USA). Using BD FACS Aria II (BD Biosciences, New Jersey, USA) cells were sorted into CD14++/CD16− (CD14, classical) and CD16 +/CD14 ++ (CD16) subpopulations. Cell purity was confirmed through flow cytometry using FACS Canto II (BD Biosciences, New Jersey, USA). 2.3. Culture of monocyte sub-populations Monocyte subpopulations were plated at a density of 70,000 cells/ well in 96-well culture plates (BD Biosciences, New Jersey, USA) and cultured overnight in RPMI-1640 complete culture medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamycin and 10% FBS (Gibco, Massachusetts, USA). 2.4. IL-4 stimulation Adherent monocytes and monocyte subpopulations were left untreated or were incubated with various concentrations of

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recombinant human IL-4 (CellGro, Freiburg, Germany) ranging from 1 to 50 ng/ml in RPMI-1640 complete culture medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamycin and 10% FBS and cultured for various time points from 24 h to seven days.

2.5. 11Β-HSD1 activity 11β-HSD1 activity was measured in terms of conversion of cortisone to cortisol by the HTRF cortisol assay kit (Cisbio Assays, Codolet, France). Cortisone (Sigma, Missouri, USA) was added to the culture medium 24 h prior to every time point to a final concentration of 100 nM and incubated for another 24 h. Cell culture supernatants were harvested and centrifuged and the clear supernatant was used for enzyme activity measurements by Time-Resolved Fluorescence Technology. HTRF technology is a competitive immunoassay in which native cortisol produced by cells and d2-labeled cortisol compete for binding to a monoclonal anti-cortisol antibody labeled with europium-Cryptate. The specific signal is inversely proportional to the concentration of cortisol in the calibrator or in the sample. The HTRF kit was used according to manufacturer's instruction and the fluorescence signals were read on a microplate reader (Victor X, Perkin Elmer, Massachusetts, USA) with an excitation filter at 340 nm and emission filters at 615 nm and 665 nm. The direct involvement of 11β-HSD1 in the conversion of cortisone to cortisol was tested by co-incubating monocytes and monocyte subpopulations with IL-4 (15 ng/ml) and a known inhibitor of 11βHSD1, Carbenoxolone [13,14] in concentrations ranging from 0.1 to 20 μM for 48 h. The fold response of 11β-HSD1 activity to IL-4 stimulation in each individual was calculated using the following formula: Fold increase = cortisol produced by IL-4 + cortisone stimulated cells / cortisol produced by cortisone stimulated cells.

2.6. 11β-HSD1 expression Gene expression analysis was performed on lysates of adherent cells obtained after collecting the supernatants for enzyme activity using Trizol reagent (Life technologies, California, USA). Total RNA was extracted from Trizol homogenates according to manufacturer's instruction. To remove potential contamination by genomic DNA, total RNA was treated with Turbo DNA-free kit (Life Technologies, California, USA). Reverse transcription was performed using the iScript cDNA synthesis kit (Bio-Rad, CA, USA) according to manufacturer's instruction. Gene expression was determined by real time RT-PCR using Sybr Green performed with the following primer sequences: HSD1 sense, 5′-AAGCAGAGCAATGGAAGCAT-3′; antisense, 5′-GAAGAACCCATCCAAAGCAA-3′; TATA box binding protein (TBP) and HPRT gene were chosen as the endogenous controls to normalize target genes: TBP sense, 5′-TGTATCCACAGTGAATCTTGG3′; TBP antisense, 5′-ATGATTACCGCAGCAAACC-3′ and HPRT sense, 5′-TGACACTGGCAAAACAATGCA; antisense, 5′-GGTCCTTTTCACCAG CAAGCT-3′. The specificity of the Sybr green fluorescence was tested by plotting fluorescence as a function of temperature to generate a melting curve of the amplicon. RT-PCR reactions were performed in 10 μl, containing 5 μl sso advanced sybergreen supermix (Bio-Rad, CA, USA), 15 ng of cDNA template and 200 nM of forward and reverse primer. The condition for PCR was set up as follows: an initial activation step at 95 °C for 30 s, followed by 40 cycles of denaturing at 95 °C for 5 s, annealing at 60 °C for 20 s. Amplifications were performed using a CFX connect Real-Time PCR detection system. Each sample was amplified in triplicate and for each procedure negative controls without template were included. Amplification efficiency was calculated for each assay by a standard curve made out of four serial dilution of a pool of cDNA samples of all time points. Comparative quantification of gene expression was determined using Pfaffl's efficiency corrected calculation.

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Fig. 1. 11β-HSD1 activity in IL-4 stimulated and unstimulated monocytes. Each graph represents data as Mean (SD) in an individual buffy coat. Control- incubated with cortisone (100 nM) alone for 24 h. IL-4-incubated with cortisone (100 nM) for 24 h and IL-4 (15 ng/ml) from 24 h to 7 days.

2.7. Analysis of CD14 and CD 16 polarization by flow cytometry Cells collected after 48 h of stimulation with IL-4 were stained for 20 min in the dark with a mix of antibodies for measuring markers of M1/M2 polarization. Mix of antibodies included the following: FITC conjugated anti-CD14 antibody (Clone MφP9, BD Biosciences, New Jersey, USA), PerCPCy5.5 conjugated anti-CD16 antibody (Clone, 3G8, BD Biosciences, New Jersey, USA), APC-H7 conjugated CD45 (Clone, 2D1, BD Biosciences, New Jersey) APC conjugated mouse anti-CD206 (Clone 19.2, BD Biosciences, New Jersey, USA), PE conjugated anti-CD163 (clone 9F5, BD Biosciences, New Jersey, USA), V450 conjugated HLADR (clone L243, BD Biosciences, New Jersey, USA) and PE-Cy™7 conjugated anti-CD64 antibody (Clone 10.1, BD Biosciences, New Jersey, USA). Expression of CD206 and CD163 proteins on the membrane surface are considered as markers for polarization of monocytes towards the M2 phenotype and the expression of CD64 and HLA-DR proteins are considered as markers for polarization of monocytes towards the M1 phenotype. The viability of the monocyte subpopulations was assessed by Annexin-V/PI assay following manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany), and analyzed by flow cytometry [15]. Surface proteins were measured using a FACSCanto II flow cytometer. At least 10,000 events were acquired for each condition.

2.8. Statistical analysis Differences in enzyme activity were analyzed by ANOVA and then Student's t-test. One-way Anova was used to evaluate phenotypical

changes among different treatments. Gene expression differences were analyzed by Student's t-test. All statistics were carried using the statistical program GraphPAD InStat, Software, USA. 3. Results 3.1. 11β-HSD1 activity and expression in cultured monocytes Freshly isolated monocytes show very little 11β-HSD1 activity (Fig. 1), but when maintained in culture, they spontaneously differentiate to macrophages and exhibit some enzyme activity. As expected from previous studies [3], monocytes showed a continuous increase in 11β-HSD1 activity till day 7. Monocytes stimulated with IL-4 showed a dose dependent increase in 11β-HSD1 activity, with the highest value at 15 ng/ml (data not shown). Hence, we selected 15 ng/ml as the concentration for our future experiments. Compared to unstimulated cells, IL-4 stimulation (15 ng/ml) showed a significant increase in 11β-HSD1 activity as early as 48–72 h of stimulation (Fig. 1). Based on this observation we selected 48 h of incubation with IL-4 as the time-point for our future studies. Monocytes isolated from buffy coats of three individuals were incubated separately with or without IL-4. All three cultures of monocytes tested showed a similar enzyme activity pattern, with high variability in the values between them. IL-4 stimulation for 48 h led to a 20.8 ± 12 (SD) fold increase in the concentration of cortisol produced when compared to that of the unstimulated monocytes in the three buffy coats tested respectively (Fig. 1a, b, c). Our results showed that 11β-HSD1 activity is present in unstimulated monocytes after 5 days of culture and it

Fig. 2. Direct involvement of 11β-HSD1 in cortisone conversion. a) Co-incubation of monocytes with IL-4, cortisone and carbenoxolone in concentrations ranging from 0.1 to 20 μM for 48 h. b) Co-incubation of CD14 monocytes with IL-4, cortisone and carbenoxolone (20 μM) for 48 h. c) Co-incubation of CD16 monocytes with IL-4, cortisone and carbenoxolone (20 μM) for 48 h. CTX- cortisone (100 nM), IL-4 (15 ng/ml), CBX-carbenoxolone (0.1 μM, 1 μM, 10 μM and 20 μM). Data are represented as Mean (SD) from 2 buffy coats.

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Fig. 3. Sorting PBMCs to obtain CD14 and CD16 monocyte subsets. Representative gating strategy for purifying and separating the two subpopulations is represented from left to right. In the first step, the population of monocytes were morphologically separated in a plot of FSC versus SSC. The singlets were gated, while cell aggregates were ignored. CD33+ cells alone were gated to avoid the lymphocyte population. A negative gate was applied to remove CD123+/CD16- cells, which include the dendritic cells. Cells were finally separated into CD14++/CD16(CD14, classical) and CD16+/CD14++ (CD16) subpopulations. a) Debris free cells b) Gating of single cells c) Distribution of CD33+ cells d) Distribution of CD123+/CD16- cells e) Distribution of CD14++/CD16- (CD14) and CD16+/CD14++ (CD16) subpopulations. Figure is representative of the 17 subjects analyzed.

is tremendously enhanced and anticipated to 2 days when incubated with IL-4. Gene expression studies showed a similar pattern as that of enzyme activity. As observed in case of enzyme activity, IL-4

stimulation (15 ng/ml) showed a significant increase in 11β-HSD1gene expression as early as 24 h, with a continuous increase up to 72 h of stimulation (Supplementary Fig. 1a).

Fig. 4. Effect of IL-4 on monocyte subpopulations. a) CD14 and CD16 cells stimulated with and without IL-4. CD14 CTX: 6.94 ± 7.96 (SD), CD14 CTX + IL-4: 52.47 ± 28.19 (SD), CD16CTX: 18.63 ± 21.18 (SD), CD16 CTX + IL-4: 31.32 ± 26.99 (SD).CTX-cortisone. b) Fold increase of cortisol production values after IL-4 stimulation. Mean fold of stimulation values of CD14: 13.85 ± 12.24 (SD) and CD16: 3.412 ± 4.052 (SD). Data are represented as Means from 17 subjects. The level of significance was set at *-P ≤ 0.001, #-P = 0.075, $-P ≤ 0.001 between indicated groups.

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Fig. 5. Cell viability analysis in IL-4 stimulated monocyte subsets. a) Measurement of percentage cell viability in CD14 cells stimulated with cortisone alone: 93.7% ± 0.28 (SD) b) Measurement of percentage cell viability in CD14 cells stimulated with IL-4 and cortisone: 89.55% ± 2.8 (SD) c) Measurement of percentage cell viability in CD16 cells stimulated with cortisone alone: 70.4 ± 4.1 (SD) d) Measurement of percentage cell viability in CD16 cells stimulated with IL-4 and cortisone: 63.8 ± 2.9 (SD). In all the figures above, cells in the lower left quadrant (annexinVneg-PIneg) represent living cells. Figure is representative of the two buffy coats tested.

To ensure that the enzyme 11β-HSD1 is the one facilitating the conversion of cortisone to cortisol, co-incubation of IL-4 stimulated monocytes with a known inhibitor of 11β-HSD1, carbenoxolone was performed. The results showed a dose dependent decrease in the concentration of cortisol measured, with the lowest value obtained at 20 μM of carbenoxolone (Fig. 2a). Based on these results from unsorted monocytes, we selected 20 μM as the concentration for our experiments in sorted cells. 3.2. Are both monocyte subpopulations, CD14 and CD16 responding to IL-4 stimulation? 3.2.1. Distribution of CD14 and CD16 on peripheral blood monocytes CD14 and CD16 monocytes were isolated from buffy coats. The percentage of CD14 and CD16 populations varied between individuals and the total mean percentage of CD14 and CD16 cells in PBMCs was 20.73 ± 6.4 (SD) % and 3.43 ± 2.39 (SD) % respectively. Purity of CD14 and CD16 cells were determined in each individual, obtaining a mean of 99.2 ± 3.2 (SD) % and 97.5 ± 2.7 (SD) % for CD14 and CD16, respectively (Fig. 3).

3.2.2. 11β-HSD1 activity of monocyte subpopulation populations Taken together, after 48 h of incubation in the presence of IL-4 (15 ng/ml), CD14 cells showed a higher 11β-HSD1 activity than the unstimulated cells (P ≤ 0.001, Fig. 4a). This increase was also reflected in the 11β-HSD1 gene expression in IL-4 stimulated CD14 cells assessed at the same point (P ≤ 0.01, Supplementary Fig. 1b). CD16 cells also showed a higher mean value of activity after incubation with IL-4, but this difference was not statistically significant (Fig. 4a). The fold response of 11β-HSD1 activity to IL-4 stimulation in CD14 and CD16 in each individual is shown in Fig. 4b. CD14 cells showed a mean fold of stimulation of 13.85 ± 12.24 (SD) while the fold increase of CD16 was 3.412 ± 4.052 (SD). There was a statistically significant difference between the mean fold of stimulation of the CD14 and CD16 cells (P ≤ 0.001). As observed in unsorted monocytes, both monocyte sub-populations showed a significant decrease in the concentration of cortisol measured when co-incubated with carbenoxolone (20 μM) indicative of the direct involvement of 11β-HSD1 enzyme in the conversion of cortisone to cortisol (Fig. 2b, c).

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Fig. 6. M1/M2 marker expression after IL-4 stimulation. a) Measurement of M2 marker CD206 in CD14 and CD16 cells after three days of culture with cortisone alone or IL-4 + cortisone. b) Measurement of M2 marker CD163 in CD14 and CD16 cells after three days of culture with cortisone alone or IL-4 + cortisone. c) Measurement of M1 marker HLA-DR in CD14 and CD16 cells after three days of culture with cortisone alone or IL-4 + cortisone. d) Measurement of M1 marker CD64 in CD14 and CD16 cells after three days of culture with cortisone alone or IL4 + cortisone *-(P ≤ 0.05). Figures represent Mean ± SEM of results from five buffy coats tested. CTX-cortisone (100 nM), IL-4 (15 ng/ml).

3.3. Are both monocyte subpopulations becoming M2 macrophages? It is accepted that monocytes stimulated with IL-4 for 7 days become M2 macrophages and express 11β-HSD1 gene [16]. Since we observed that 11β-HSD1 gene activity was increased in CD14 as soon as the third day of IL-4 stimulation, we next questioned if the M2 phenotype was a prerequisite for 11β-HSD1 gene expression and analyzed monocyte subpopulation phenotypes according to the expression of M1 and M2 markers by flow cytometry. After 48 h of IL-4 and 24 h of cortisone stimulation the viability of CD14 and CD16 cells was 89.55 ± 2.8 (SD) % (Fig. 5b) and 63.8 ± 2.9 (SD) % (Fig. 5d) respectively and that of unstimulated cells subjected to 24 h of cortisone stimulation alone was 93.7 ± 0.28 (SD) % (Fig. 5a) and 70.4 ± 4.1 (SD) % (Fig. 5c) in CD14 and CD16 cells respectively. In a separate experiment, CD16 cells stimulated with IL-4 alone showed a higher viability of 85.6% (results not shown) suggesting that cortisone stimulation might be the reason for the reduced viability of CD16 subpopulation. Our observation is supported by previous reports, which state that glucocorticoid therapy could be responsible for the selective depletion of the CD16 (CD14+ CD16+) monocyte subpopulation [17–19].

Expression of the surface proteins CD206 and CD163 was measured for checking polarization towards the M2 phenotype and expression of CD64 and HLA-DR proteins were measured for checking polarization towards the M1 phenotype. Fluorescence intensity was measured for each treatment group and results were expressed as median fluorescence intensity (rMFI). M2 markers CD206 (Fig. 6a), CD163 (Fig. 6b), and M1 marker HLA-DR (Fig. 6c) showed no significant change in both CD14 and CD16 cells when stimulated with IL-4 for three days. Expression of M1 marker CD64 showed a significant decrease (P ≤ 0.05) after IL-4 stimulation in CD14 cells (Fig. 6d, Supplementary Fig. 2), but there was no significant change in its expression in IL-4 stimulated CD16 cells (Fig. 6d). 4. Discussion Glucocorticoids such as cortisone or prednisone may work therapeutically only if they are converted to active glucocorticoids (cortisol and prednisolone) by the intracellular HSD enzymes [20]. These enzymes interconvert the active and inactive forms of glucocorticoids. The 11β-HSD1 enzyme is bidirectional, but primarily activates glucocorticoids, whereas the 11β-HSD2 enzyme is unidirectional and a powerful

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glucocorticoid inactivator. None of the circulating cells express HSD1 activity, but in activated stromal or inflammatory tissue both 11β-HSD1 and 11β-HSD2 enzymes are expressed [3,21]. Furthermore, the capacity to convert glucocorticoids to cortisone (a unique feature of 11β-HSD1) increases with the degree of inflammation [22]. 11β-HSD1 has been reported to play a role in the resolution of inflammation by macrophages and has been indicated as a novel target for therapy [23]. In the inflammatory synovium of rheumatoid arthritis, a deficiency in glucocorticoid activation has been observed as a consequence of either increased expression of 11β-HSD2 or an inadequate expression/activity of 11βHSD1 [24,25]. A number of patients have been identified with the putative 11β-HSD1-deficient state, with an impaired capacity to generate cortisol from an ingested dose of cortisone acetate, indicating a lack of 11β-HSD1 reductase activity in the liver [26,27]. Blood monocytes consist of different subpopulations of cells, which differ in size, nuclear morphology, granularity, and functionality. With proper cell surface markers monocytes can be subdivided into two main populations, i.e., the CD14 + CD16 + and the CD14 monocytes [28]. CD16 monocytes, unlike CD14, are potent producers of TNF [11, 28] and the ratio between CD14/CD16 is inversely correlated in inflammatory diseases like rheumatoid arthritis severity [29]. Also glucocorticoid therapy decreases CD16 and increases CD14 [17], all of which makes CD16 crucial players in inflammation [30]. 11β-HSD1 is potently regulated by cytokines in both immune [3] and non-immune cells [31,32,33,34,35,36,37]. For instance, HSD11b1 (encoding 11β-HSD1) is not expressed in human monocytes but is induced upon differentiation to macrophages, where its expression is further increased by the anti-inflammatory cytokines IL-4 and IL-13 [3]. Various therapeutic options available to reduce inflammation target the cytokines involved in regulating inflammation. Recombinant IL-4 has previously been investigated in preclinical models of rheumatoid arthritis, showing disease-modifying efficacy [38,39,40]. A recent study investigated the successful targeting and therapeutic activity of the immunocytokine F8-IL-4 in the mouse model of collagen-induced arthritis. When used in combination with the glucocorticoid dexamethasone, F8-IL4 was able to cure mice with established collagen-induced arthritis fully human version of F8-IL4 is currently being developed for clinical investigations [41]. IL-4 is a pleiotropic cytokine produced by activated T cells that has been discussed as a monocyte activating protein that affects the phenotype and functions of human monocytes and macrophages [42]. It has already been demonstrated that blood monocytes stimulated with IL4 showed an increase in 11β-HSD1 activity [3], but it is still unknown if all monocyte subsets respond to IL-4 induction in a similar way or if the increased 11β-HSD1 activity after exposure to IL-4 is specific to one of the monocyte subpopulations. In this study we analyzed 11βHSD1 activity in CD14 and CD16 monocyte subpopulations to determine if they respond differentially to the stimulus of IL-4. The 11βHSD1 activity in unstimulated CD14 was lower than that observed in unstimulated CD16, but a statistically significant increase was observed only in CD14 cells with a 4 folds greater activity than that observed in CD16 monocytes. These results suggest that there are no major differences in the potential of CD14 and CD16 to produce the 11β-HSD1 enzyme, but the difference becomes substantial when the cells are exposed to IL-4. Monocyte derived macrophages M1 or M2, play different roles in the inflammatory processes. Indeed, M1 macrophages are considered strongly pro-inflammatory, whereas M2 macrophages produce high amounts of anti-inflammatory molecules such as IL-10, TGFβ and IL1Ra [43]. Expression of 11β-HSD1 gene has been reported to be dependent on the macrophage phenotype since macrophages express higher gene and protein levels of the enzyme than resting monocytes (RM) and M1 macrophages, resulting in a more pronounced conversion of cortisone into active cortisol [12]. In our study we demonstrate that the varying response of CD14 and CD16 to IL-4, may be related to their differential tendency to mature towards M1 or M2, which can be

seen by the low but significant reduction of M1 markers in CD14 cells after IL-4 stimulation. But absence of increase in M2 markers after IL-4 stimulation shows that although IL-4 stimulated CD14 cells are not yet of the M2 phenotype they are already able to show an increase in 11β-HSD1 enzyme activity as early as 48 h after stimulation. The reduced ability of CD16 cells to produce 11β-HSD1 enzyme when stimulated with the anti-inflammatory cytokine IL-4 further strengthen their pro-inflammatory nature. In conclusion, in this study we confirmed the potential role of IL-4 in the induction of 11β-HSD1 in maturing monocytes. The activity of the enzyme is tremendously potentiated by the IL-4, which hampers the maturation of CD14 cells alone towards the pro-inflammatory M1. Out of the two subpopulations studied CD14 cells are the major source of HSD1 enzyme after IL-4 stimulation, independent of the M2 phenotype. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2016.12.015. Conflict of interest statement The authors declare no financial or commercial conflict of interest. Funding This work was funded by “Progetti Finalizzati”, Regione Veneto (No. 155). Acknowledgements We would like to thank Dr. Maria Teresa Scupoli for the provision of equipment and Eleonora Cappelletti for technical assistance. This study was performed (in part) in the LURM (Laboratorio Universitario di Ricerca Medica) Research Center, University of Verona. References [1] P.M. Stewart, Z.S. Krozowski, 11β-Hydroxysteroid dehydrogenase, Vitam. Horm. 57 (1999) 249–324. [2] T.Y. Zhang, X. Ding, R.A. Daynes, The expression of 11β-hydroxysteroid dehydrogenase type I by lymphocytes provides a novel means for intracrine regulation of glucocorticoid activities, J. Immunol. 174 (2005) 879–889. [3] R. Thieringer, C.B. Le Grand, L. Carbin, T.Q. Cai, B. Wong, S.D. Wright, A. Hermanowski-Vosatka, 11β-hydroxysteroid dehydrogenase type 1 is induced in human monocytes upon differentiation to macrophages, J. Immunol. 167 (2001) 30–35. [4] P.H. Hart, G.F. Vitti, D.R. Burgess, G.A. Whitty, D.S. Piccoli, J.A. Hamilton, Potential anti-inflammatory effects of interleukin-4. Suppression of human monocyte TNFα, IL-1 and PGE2 levels, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 3803–3807. [5] P. Miossec, J. Chomarat, P. Dechanet, J.F. Moreau, J.P. Roux, P. Delmas, J. Banchereau, Interleukin-4 inhibits bone resorption through an effect on osteoclasts and proinflammatory cytokines in an ex vivo model of bone resorption in rheumatoid arthritis, Arthritis Rheum. 37 (1994) 1715–1722. [6] E. Lubberts, L.A. Joosten, L. Van Den Bersselaar, M.M. Helsen, A.C. Bakker, J.B. van Meurs, F.L. Graham, C.D. Richards, W.B. van Den Berg, Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction, J. Immunol. 163 (1999) 4546–4556. [7] R.M. Clarke, A. Lyons, F. O'Connell, B.F. Deighan, C.E. Barry, N.G. Anyakoha, A. Nicolaou, M.A. Lynch, A pivotal role for interleukin-4 in atorvastatin-associated neuroprotection in rat brain, J. Biol. Chem. 283 (2008) 1808–1817. [8] T. Aprahamian, R. Bonegio, J. Rizzo, H. Perlman, D.J. Lefer, I.R. Rifkin, K. Walsh, Simvastatin treatment ameliorates autoimmune disease associated with accelerated atherosclerosis in a murine lupus model, J. Immunol. 177 (2006) 3028–3034. [9] D. Hudig, K.W. Hunter, W.J. Diamond, D. Redelman, Properties of human blood monocytes. II. Monocytes from healthy adults are highly heterogeneous within and among individuals, Cytometry B Clin. Cytom. 86 (2014) 121–134. [10] L. Ziegler-Heitbrock, Monocyte subsets in man and other species, Cell. Immunol. 289 (2014) 135–139. [11] L. Ziegler-Heitbrock, The CD14+ CD16+ blood monocytes: their role in infection and inflammation, J. Leukoc. Biol. 8 (2007) 1584–1592. [12] G. Chinetti-Gbaguidi, M.A. Bouhlel, C. Copin, C. Duhem, B. Derudas, B. Neve, B. Noel, J. Eeckhoute, P. Lefebvre, J.R. Seckl, B. Staels, Peroxisome proliferator-activated receptor ɣ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages, Arterioscler. Thromb. Vasc. Biol. 32 (2012) 677–685. [13] G.M. Coppola, P.J. Kukkola, J.L. Stanton, A.D. Neubert, N. Marcopulos, N.A. Bilci, H. Wang, H.C. Tomaselli, J. Tan, T.D. Aicher, D.C. Knorr, A.Y. Jeng, B. Dardik, R.E.

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