*6-([1,1'-Biphenyl]-4-yl)-2-hydroxy-3-methylhexanoic acid* (**9**). To a solution of compound **8** (200 mg, 0.64 mmol) in MeOH (6 mL), a 1N aqueous solution of NaOH (5 mL) was added and the reaction mixture was left stirring at room temperature for 2 days. After acidification with 1N HCl (pH 1), the reaction mixture was extracted with Et2O (3 × 20 mL). The combined organic layers were washed with H2O (2 × 10 mL) and dried over MgSO4. The solvent was removed under reduced pressure and the product was purified by column chromatography (petroleum ether 40–60 ◦C:EtOAc) (8:2). Mixture of diastereomers; Yield 64%; White solid; mp: 147–151 oC; 1H NMR (200 MHz, CD3OD): δ = 7.61–7.14 (m, 9H), 4.12–3.97 (m, 1H), 2.70–2.53 (m, 2H), 2.02–1.85 (m, 1H), 1.73–1.44 (m, 4H), 1.02–0.82 (m, 3H); 13C NMR (50 MHz, CD3OD): δ = 176.6, 176.2, 141.8, 141.2, 138.7, 128.8, 128.6, 126.8, 126.7, 126.6, 126.5, 74.5, 73.0, 37.0, 35.5, 35.4, 29.2, 15.4, 13.1; HRMS [M-H]- : 297.1492; (calculated for [C19H21O3] - 297.1496).

*tert-Butyl 5-bromohexanoate* (**12**). In a 25 mL round bottom flask, δ-hexalactone (342 mg, 3.00 mmol) and 33% HBr in AcOH (5 mL) were added and the reaction mixture was left stirring at 75 ◦C for 16 h. After completion of the reaction, the reaction mixture was extracted with CH2Cl2 (3 × 15 mL). The organic layers were collected, washed with H2O (3 × 15 mL) and dried over MgSO4. The solvent was removed under reduced pressure and the formed bromo carboxylic acid was used directly to the next step. To a solution of the bromo carboxylic acid (544 mg, 2.82 mmol) in dry CH2Cl2 (20 mL) in a pressure vessel, concentrated H2SO4 (0.45 mL) was added dropwise. The reaction mixture was cooled at −196 ◦C using liquid nitrogen and isobutylene (20 mL) was added to the solution. The reaction mixture was left stirring at room temperature for 5 d. The reaction mixture was poured to a 100 mL beaker and was left stirring for 30 min. The reaction mixture was washed with H2O (3 × 15 mL) and the organic phase was dried over MgSO4. The solvent was removed under reduced pressure and the product was purified by column chromatography (petroleum ether 40–60 ◦C:EtOAc) (9:1). Yield 83%; Yellowish oil; 1H NMR (200 MHz, CDCl3): δ = 4.12–4.02 (m, 1H), 2.23-2.14 (m, 2H), 1.79–1.62 (m, 7H), 1.39 (s, 9H); 13C NMR (50 MHz, CDCl3): δ = 172.7, 80.4, 51.1, 40.4, 34.8, 28.3, 26.6, 23.4; HRMS [M+Na]+: 273.0467; (calculated for [C10H19BrNaO2] + 273.0461).

General procedure for the synthesis of 2-hydroxy esters **15a,b**.

To a stirred solution of compound **9** or **13** (0.38 mmol) in THF (2.4 mL), a 20% aqueous solution of Cs2CO3 (124 mg, 0.38 mmol) was added (pH 9) and the reaction mixture was left stirring for 20 min at room temperature. The organic solvent was evaporated under reduced pressure and the residue was dissolved in DMF (6 mL). *tert*-Butyl ester **14** or **12** (0.46 mmol) was added and the reaction mixture was left stirring under reflux for 72 h to 168 h. Then, H2O (10 mL) was added and the reaction mixture was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over MgSO4 and the solvent was removed under reduced pressure. The product was purified by column chromatography (petroleum ether 40–60 ◦C:EtOAc) (8:2).

*5-(tert-Butoxy)-5-oxopentyl 6-([1,1'-biphenyl]-4-yl)-2-hydroxy-3-methylhexanoate* (**15a**). Mixture of diastereomers; Yield 35%; Colourless oil; 1H NMR (200 MHz, CDCl3): δ = 7.64–7.17 (m, 9H), 4.25–4.05 (m, 3H), 2.72-2.57 (m, 2H), 2.31–2.16 (m, 2H), 2.05-1.52 (m, 8H), 1.44 (s, 9H), 1.01 (d, *J* = 6.9 Hz, 1.5H), 0.84 (d, *J* = 6.9 Hz, 1.5H); 13C NMR (50 MHz, CDCl3): δ = 175.5, 175.1, 172.8, 141.9, 141.8, 141.3, 138.9, 134.1, 129.1, 129.0, 128.9, 127.2, 123.4, 80.6, 75.1, 73.4, 65.5, 65.4, 37.5, 36.9, 35.9, 35.1, 35.0, 33.1, 30.8, 29.6, 29.3, 28.3, 28.1, 21.7, 16.1, 13.7; HRMS [M+Na]+: 477.2608; (calculated for [C28H38NaO5] + 477.2611).

*6-(tert-Butoxy)-6-oxohexan-2-yl 6-([1,1'-biphenyl]-4-yl)-2-hydroxyhexanoate*(**15b**). Mixture of diastereomers; Yield 37%; Colourless oil; 1H NMR (200 MHz, CDCl3): δ = 7.64–7.17 (m, 9H), 5.02–4.90 (m, 1H), 4.18–4.07 (m, 1H), 2.98–2.54 (m, 4H), 2.29–2.10 (m, 2H), 1.90–1.50 (m, 8H), 1.43 (s, 9H), 1.28-1.19 (m, 3H); 13C NMR (50 MHz, CDCl3): δ = 175.2, 172.8, 172.7, 141.8, 141.7, 141.3, 138.9, 129.0, 128.9, 127.3, 127.2, 80.5, 72.6, 70.7, 70.5, 35.7, 35.6, 35.2, 34.6, 34.5, 31.5, 31.4, 28.3, 24.8, 24.6, 21.0, 20.1, 20.0; HRMS [M+Na]+: 477.2614; (calculated for [C28H38NaO5] + 477.2611).

General procedure for the synthesis of 2-oxoesters **16a,b**.

To a solution of compound **15** (0.13 mmol) in dry CH2Cl2 (2.0 mL) was added Dess-Martin periodinane (72 mg, 0.17 mmol) and the reaction mixture was left stirring at room temperature for 2 to 4 h. Then, a 10% aqueous solution of Na2S2O3 (5 mL) was added and the reaction mixture was extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were mixed and dried over MgSO4 and the solvent was removed under reduced pressure. The product was purified by column chromatography (petroleum ether 40–60 ◦C:EtOAc) (8:2).

*5-(tert-Butoxy)-5-oxopentyl 6-([1,1'-biphenyl]-4-yl)-3-methyl-2-oxohexanoate* (**16a**). Yield 75%; Colourless oil; 1H NMR (200 MHz, CDCl3): δ = 7.63–7.19 (m, 9H) 4.24 (t, *J* = 6.3 Hz, 2H), 3.31–3.15 (m, 1H), 2.65 (t, *J* = 7.2 Hz, 2H), 2.25 (t, *J* = 6.9 Hz, 2H), 1.89-1.56 ( m, 8H), 1.44 (s, 9H), 1.15 (d, *J* = 7.0 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ = 198.1, 172.7, 162.1, 141.2, 139.0, 129.0, 128.9, 127.3, 127.2, 127.1, 80.6, 66.0, 42.3, 35.6, 35.1, 31.7, 29.0, 28.3, 28.0, 21.6, 15.4; HRMS [M+Na]+: 475.2434; (calculated for [C28H36NaO5] + 475.2455).

*6-(tert-Butoxy)-6-oxohexan-2-yl 6-([1,1'-biphenyl]-4-yl)-2-oxohexanoate* (**16b**). Yield 77%; Yellowish oil; 1H NMR (200 MHz, CDCl3): δ = 7.63–7.17 (m, 9H), 5.09–4.96 (m, 1H), 2.92–2.78 (m, 2H), 2.75–2.59 (m, 2H) 2.29–2.12 (m, 2H), 1.83–1.52 (m, 8H), 1.48–1.22 (m,12H); 13C NMR (50 MHz, CDCl3): δ = 194.9, 172.7, 161.1, 141.3, 139.0, 129.0, 128.9, 127.3, 127.2, 80.5, 73.7, 39.4, 35.4, 35.2, 35.1, 30.9, 28.3, 22.8, 21.0, 19.9; HRMS [M+Na]+: 475.2456; (calculated for [C28H36NaO5] + 475.2455).

General procedure for the synthesis of 2-oxoesters **17a,b**.

A solution of *tert*-butyl ester **16** (0.13 mmol) in dry CH2Cl2 (3.0 mL) and TFA (3.0 mL) was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and then CH2Cl2 (5 mL) was added and re-evaporated twice. The product was purified by column chromatography (petroleum ether 40–60 ◦C:EtOAc) (8:2).

*5-((6-([1,1'-Biphenyl]-4-yl)-3-methyl-2-oxohexanoyl)oxy)pentanoic acid* (**17a**) (GK587). Yield 77%; Colourless oil; 1H NMR (200 MHz, CDCl3): δ = 7.63–7.16 (m, 9H), 4.25 (t, *J* = 6.3 Hz, 2H), 3.34–3.12 ( m, 1H), 2.65 (t, *J* = 7.2 Hz, 2H), 2.39 (t, *J* = 6.8 Hz, 2H),1.91–1.54 (m, 8H), 1.15 (d, *J* = 7.0 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ = 198.0, 179.4, 162.0, 141.2, 139.0, 129.0, 128.9, 127.3, 127.2, 65.9, 42.3, 35.6, 33.5, 31.6, 29.0, 27.9, 21.2, 15.4; HRMS [M-H]- : 395.1854; (calculated for [C24H27O5] - 395.1864).

*5-((6-([1,1'-Biphenyl]-4-yl)-2-oxohexanoyl)oxy)hexanoic acid* (**17b**) (GK639). Yield 87%; Colourless oil; 1H NMR (200 MHz, CDCl3): δ = 7.62-7.17 (m, 9H), 5.10–4.94 (m, 1H), 2.94–2.77 (m, 2H), 2.73–2.57 (m, 2H), 2.44-2.28 (m, 2H),1.85–1.52 (m, 8H), 1.30 (d, *J* = 7.0 Hz, 3H); 13C NMR (50 MHz, CDCl3): δ = 194.9, 179.5, 161.1, 141.3, 139.0, 129.0, 128.9, 127.3, 127.3, 127.2, 73.6, 39.4, 35.4, 35.0, 33.7, 30.9, 22.8, 20.6, 19.9; HRMS [M-H]- : 395.1857; (calculated for [C24H27O5] - 395.1864).

#### *2.2. Plasma Stability Studies*

For plasma stability, LC-MS studies were performed with an ABSciex Triple TOF 4600 (ABSciex, Darmstadt, Germany) combined with a micro-LC Eksigent (Eksigent, Darmstadt, Germany) and an autosampler set at 5 ◦C and a thermostated column compartment. Electrospray ionization (ESI) in negative mode was used for the MS experiments. The data acquisition method consisted of a TOF-MS full scan *m*/*z* 50–850 Da and several IDA-TOF-MS/MS (Information Dependent Acquisition) product ion scans using 40 V Collision Energy (CE) with 15 V (Collision Energy Spread) CES used for each candidate ion in each data acquisition cycle (1091). Halo C18 2.7 µm, 90 Å, 0.5 × 50 mm2 from Eksigent was used as a column and the mobile phase consisted of a gradient (A: acetonitrile/0.01% formic acid/isopropanol 80/20 v/v; B: H2O/0.01% formic acid). The elution gradient adopted started with 5% of phase B for 0.5 min, gradually increasing to 98% in the next 7.5 min. These conditions were kept constant for 0.5 min, and then the initial conditions (95% solvent B, 5% solvent A) were restored within 0.1 min to re-equilibrate the column for 1.5 min for the next injection (flow rate 55 µL/min). The data acquisition was carried out with MultiQuant 3.0.2 and PeakView 2.1 from AB SCIEX (ABSciex, Darmstadt, Germany).

## *2.3. In vitro PLA2 Activity Assay*

Group specific PLA2 assays were employed to determine the inhibitory activity using a lipidomics-based mixed micelle assay as previously described [4,25]. The substrate for each enzyme consisted of 100 µM PAPC (except for GIVA cPLA2 as noted), 400 µM of C12E8 surfactant, and 2.5 µM of 17:0 LPC internal standard. For GIVA cPLA2, the total phospholipid concentration (100 µM) consisted of 97 µM PAPC and 3 µM of PI(4,5)P2 which enhances the activity of the enzyme. A specific buffer was prepared to achieve optimum activity for each enzyme. The buffer for GIVA cPLA2 contained 100 mM HEPES pH 7.5, 90 µM CaCl2, and 2 mM DTT. For GVIA iPLA2, the buffer consisted of 100 mM HEPES pH 7.5, 2 mM ATP, and 4 mM DTT. Finally, the buffer for GV sPLA2 contained 50 mM Tris-HCl pH 8.0 and 5 mM CaCl2. The enzymatic reaction was performed in a 96 well-plate using a Benchmark Scientific H5000-H MultiTherm heating shaker for 30 min at 40 ◦C. Each reaction was quenched with 120 µL of methanol/acetonitrile (80/20, v/v), and the samples were analyzed using a HPLC-MS system. A Shimadzu SCL-10A system controller with two LC-10AD liquid pumps (Shimadzu Corp.,Kyoto Kyoto, Japan) connected to a column controller instrument (Analytical Sales & Products, Inc, Flanders NJ, USA), and a CTC Analytics PAL autosampler platform (Leap Technologies, NC, USA) were used for HPLC analysis. An AB Sciex 4000 QTRAP triple quadrupole/linear ion trap hybrid mass spectrometer (AB Sciex LLC, Framingham MA, USA) was used for MS analysis. A blank experiment, which did not contain enzyme, was also included for each substrate to determine the non-enzymatic hydrolysis product and to detect any changes in the intensity of the 17:0 LPC internal standard.

#### **3. Results and Discussion**

#### *3.1. Design and Synthesis of Inhibitors*

We assumed that in human plasma, it is likely that the oxoester functionality is hydrolyzed by an esterase. We hypothesized that if a small organic functionality, such as a methyl group, was added, either on the α-carbon to the oxoester functionality or on the α-carbon to the ester oxygen, it would decrease the rate of destruction of these compounds. The methyl group is the smallest alkyl group and has played a beneficial role in drug design by often constituting the problem-solving key to lead

optimization [26]. Mainly, the PharmacoKinetic (PK) and PharmacoDynamic (PD) properties of a compound can be modified by the addition of the methyl group, where in many cases an increase in the selectivity and potency of the pharmaceutical agent has resulted. *Biomolecules* **2020**, *10*, x 7 of 12 of a compound can be modified by the addition of the methyl group, where in many cases an increase in the selectivity and potency of the pharmaceutical agent has resulted. *Biomolecules* **2020**, *10*, x 7 of 12 of a compound can be modified by the addition of the methyl group, where in many cases an

GK452 (**1a**, Figure 1) was one of the most potent 2-oxoester inhibitors of GIVA cPLA2. Thus, we envisaged that the two methylated derivatives of this compound shown in Figure 2 should result in an increased metabolic stability. GK452 (**1a**, Figure 1) was one of the most potent 2-oxoester inhibitors of GIVA cPLA2. Thus, we envisaged that the two methylated derivatives of this compound shown in Figure 2 should result in an increased metabolic stability. increase in the selectivity and potency of the pharmaceutical agent has resulted. GK452 (**1a**, Figure 1) was one of the most potent 2-oxoester inhibitors of GIVA cPLA2. Thus, we envisaged that the two methylated derivatives of this compound shown in Figure 2 should result in an increased metabolic stability.

**Figure 2.** Design of α-methyl derivatives of **GK452** inhibitor of GIVA cPLA2. **Figure 2.** Design of α-methyl derivatives of **GK452** inhibitor of GIVA cPLA2 . **Figure 2.** Design of α-methyl derivatives of **GK452** inhibitor of GIVA cPLA2.

The synthetic strategy for how to insert the methyl group in the desired positions (left or right of the oxoester) was, firstly, with a lithium diisopropylamide (LDA) treatment followed by methylation for the left addition, and by a coupling reaction of an intermediate α-hydroxy carboxylic acid to a methylated secondary bromo *tert*-butyl ester for the right addition. The synthetic strategy for how to insert the methyl group in the desired positions (left or right of the oxoester) was, firstly, with a lithium diisopropylamide (LDA) treatment followed by methylation for the left addition, and by a coupling reaction of an intermediate α-hydroxy carboxylic acid to a methylated secondary bromo *tert*-butyl ester for the right addition. The synthetic strategy for how to insert the methyl group in the desired positions (left or right of the oxoester) was, firstly, with a lithium diisopropylamide (LDA) treatment followed by methylation for the left addition, and by a coupling reaction of an intermediate α-hydroxy carboxylic acid to a methylated secondary bromo *tert*-butyl ester for the right addition.

The synthesis of the needed α-hydroxy β-methyl carboxylic acid **9**, bearing a biphenyl scaffold on the left, is described in Scheme 1. The first reaction was an α-substitution using an LDA method, hexamethylphosphoramide (HMPA) to help stabilize the formed enolate and CH3I as the electrophile [27], giving the α-methyl carboxylic acid **4** in a high yield. Then, the carboxylic moiety was reduced to an alcohol by converting it into the corresponding mixed anhydride and finally reducing it with NaBH4 in the presence of MeOH [28]. Alcohol **5**, which was obtained in a high yield, was next oxidized using PCC to aldehyde **6**, which after reaction with KCN gave cyanohydrin **7** in an excellent yield. Acidic methanolysis by treatment with freshly prepared 6N HCl in MeOH led to the corresponding hydroxy methyl ester **8** in a good yield, which finally, after saponification, afforded the desired α-hydroxy carboxylic acid **9**. The synthesis of the needed α-hydroxy β-methyl carboxylic acid **9**, bearing a biphenyl scaffold on the left, is described in Scheme 1. The first reaction was an α-substitution using an LDA method, hexamethylphosphoramide (HMPA) to help stabilize the formed enolate and CH3I as the electrophile [27], giving the α-methyl carboxylic acid **4** in a high yield. Then, the carboxylic moiety was reduced to an alcohol by converting it into the corresponding mixed anhydride and finally reducing it with NaBH4 in the presence of MeOH [28]. Alcohol **5**, which was obtained in a high yield, was next oxidized using PCC to aldehyde **6**, which after reaction with KCN gave cyanohydrin **7** in an excellent yield. Acidic methanolysis by treatment with freshly prepared 6N HCl in MeOH led to the corresponding hydroxy methyl ester **8** in a good yield, which finally, after saponification, afforded the desired α-hydroxy carboxylic acid **9**. The synthesis of the needed α-hydroxy β-methyl carboxylic acid **9**, bearing a biphenyl scaffold on the left, is described in Scheme 1. The first reaction was an α-substitution using an LDA method, hexamethylphosphoramide (HMPA) to help stabilize the formed enolate and CH3I as the electrophile [27], giving the α-methyl carboxylic acid **4** in a high yield. Then, the carboxylic moiety was reduced to an alcohol by converting it into the corresponding mixed anhydride and finally reducing it with NaBH4 in the presence of MeOH [28]. Alcohol **5**, which was obtained in a high yield, was next oxidized using PCC to aldehyde **6**, which after reaction with KCN gave cyanohydrin **7** in an excellent yield. Acidic methanolysis by treatment with freshly prepared 6N HCl in MeOH led to the corresponding hydroxy methyl ester **8** in a good yield, which finally, after saponification, afforded the desired α-hydroxy carboxylic acid **9**.

**Scheme 1.** Synthesis of 2-hydroxy acid **9**. (**a**) (i) LDA, dry THF (ii) HMPA, (iii) CH3I; (**b**) (i) CH3CH2OCOCl, Et3N, dry THF, (ii) NaBH4, MeOH; (**c**) iodobenzene diacetate, TEMPO (10 mol%), dry CH2Cl2; (**d**) aq. sol. NaHSO3, CH2Cl2, (ii) KCN, H2O; (**e**) 6N HCl/MeOH; (**f**) 1N NaOH, MeOH. **Scheme 1.** Synthesis of 2-hydroxy acid **9**. (**a**) (i) LDA, dry THF (ii) HMPA, (iii) CH3I; (**b**) (i) CH3CH2OCOCl, Et3N, dry THF, (ii) NaBH4, MeOH; (**c**) iodobenzene diacetate, TEMPO (10 mol%), dry CH2Cl2; (**d**) aq. sol. NaHSO3, CH2Cl2, (ii) KCN, H2O; (**e**) 6N HCl/MeOH; (**f**) 1N NaOH, MeOH. **Scheme 1.** Synthesis of 2-hydroxy acid **9**. (**a**) (i) LDA, dry THF (ii) HMPA, (iii) CH3 I; (**b**) (i) CH3CH2OCOCl, Et3N, dry THF, (ii) NaBH4 , MeOH; (**c**) iodobenzene diacetate, TEMPO (10 mol%), dry CH2Cl2 ; (**d**) aq. sol. NaHSO3 , CH2Cl2 , (ii) KCN, H2O; (**e**) 6N HCl/MeOH; (**f**) 1N NaOH, MeOH.

For the synthesis of *tert*-butyl ester **12**, acidic bromination of commercially available delta-hexalactone **10**, using 33% HBr in AcOH [29], produced the intermediate bromo carboxylic

For the synthesis of *tert*-butyl ester **12**, acidic bromination of commercially available delta-hexalactone **10**, using 33% HBr in AcOH [29], produced the intermediate bromo carboxylic

For the synthesis of *tert*-butyl ester **12**, acidic bromination of commercially available delta-hexalactone **10**, using 33% HBr in AcOH [29], produced the intermediate bromo carboxylic acid **11** in almost quantitative yield. Next step was the protection of the carboxyl group using isobutylene in the presence of c. H2SO4, leading to *tert*-butyl ester **12** in a high yield (Scheme 2). *Biomolecules* **2020**, *10*, x 8 of 12 *Biomolecules* **2020**, *10*, x 8 of 12

**Scheme 2.** Synthesis of bromo *tert*-butyl ester **12**. (**a**) 33% HBr in AcOH; (**b**) isobutylene, c.H2SO4, dry CH2Cl2. **Scheme 2.** Synthesis of bromo *tert*-butyl ester **12**. (**a**) 33% HBr in AcOH; (**b**) isobutylene, c.H2SO4 , dry CH2Cl2 . c.H2SO4, dry CH2Cl2.

**Scheme 2.** Synthesis of bromo *tert*-butyl ester **12**. (**a**) 33% HBr in AcOH; (**b**) isobutylene,

After having synthesized the needed acid **9** and the appropriate hydroxy acid **13**, as previously described [23], a coupling reaction was performed by treatment with Cs2CO3 and the corresponding bromo *tert*-butyl esters (methylated or not) (Scheme 3). The coupling products **15a** and **15b** were produced in moderate yields 35–37%. For this step, it was observed that, when *tert*-butyl ester **12**  was used, an increased reaction time was needed. Oxidation using Dess-Martin periodinane in dry CH2Cl2 gave oxoesters **16a** and **16b** in high yields. Final deprotection of the *tert*-butyl ester by using 50% TFA in dry CH2Cl2 led to the desired products **17a** (GK587) and **17b** (GK639) methylated After having synthesized the needed acid **9** and the appropriate hydroxy acid **13**, as previously described [23], a coupling reaction was performed by treatment with Cs2CO3 and the corresponding bromo *tert*-butyl esters (methylated or not) (Scheme 3). The coupling products **15a** and **15b** were produced in moderate yields 35–37%. For this step, it was observed that, when *tert*-butyl ester **12** was used, an increased reaction time was needed. Oxidation using Dess-Martin periodinane in dry CH2Cl2 gave oxoesters **16a** and **16b** in high yields. Final deprotection of the *tert*-butyl ester by using 50% TFA in dry CH2Cl2 led to the desired products **17a** (GK587) and **17b** (GK639) methylated derivatives. After having synthesized the needed acid **9** and the appropriate hydroxy acid **13**, as previously described [23], a coupling reaction was performed by treatment with Cs2CO3 and the corresponding bromo *tert*-butyl esters (methylated or not) (Scheme 3). The coupling products **15a** and **15b** were produced in moderate yields 35–37%. For this step, it was observed that, when *tert*-butyl ester **12**  was used, an increased reaction time was needed. Oxidation using Dess-Martin periodinane in dry CH2Cl2 gave oxoesters **16a** and **16b** in high yields. Final deprotection of the *tert*-butyl ester by using 50% TFA in dry CH2Cl2 led to the desired products **17a** (GK587) and **17b** (GK639) methylated derivatives.

**Scheme 3.** Synthesis of 2-oxoesters. (**a**) (i) Cs2CO3, THF, H2O (pH 9); (ii) DMF, reflux, 24–72 h; (**b**) **Scheme 3.** Synthesis of 2-oxoesters. (**a**) (i) Cs2CO3, THF, H2O (pH 9); (ii) DMF, reflux, 24–72 h; (**b**) Dess Martin, dry CH2Cl2, r.t., 3 h; (**c**) 50% TFA, dry CH2Cl2, r.t., 2 h. **Scheme 3.** Synthesis of 2-oxoesters. (**a**) (i) Cs2CO3 , THF, H2O (pH 9); (ii) DMF, reflux, 24–72 h; (**b**) Dess Martin, dry CH2Cl2 , r.t., 3 h; (**c**) 50% TFA, dry CH2Cl2 , r.t., 2 h.

#### Dess Martin, dry CH2Cl2, r.t., 3 h; (**c**) 50% TFA, dry CH2Cl2, r.t., 2 h. *3.2. Plasma Stability Studies 3.2. Plasma Stability Studies*

as well.

as well.

*3.2. Plasma Stability Studies*  The in vitro stability of 2-oxoesters **17a** and **17b**, as well as inhibitor GK452 for comparison, was studied in human plasma. The reactions were initiated by the addition of test compound to a preheated plasma solution to yield a final concentration of 1 mg/L [24]. Samples (50 μL) were obtained and after the appropriate treatment were analyzed by LC-HRMS/MS (ABSciex, Darmstadt, The in vitro stability of 2-oxoesters **17a** and **17b**, as well as inhibitor GK452 for comparison, was studied in human plasma. The reactions were initiated by the addition of test compound to a preheated plasma solution to yield a final concentration of 1 mg/L [24]. Samples (50 μL) were obtained and after the appropriate treatment were analyzed by LC-HRMS/MS (ABSciex, Darmstadt, Germany) and the results are shown in Figure 3 (see, also Supplementary Material). The in vitro stability of 2-oxoesters **17a** and **17b**, as well as inhibitor GK452 for comparison, was studied in human plasma. The reactions were initiated by the addition of test compound to a preheated plasma solution to yield a final concentration of 1 mg/L [24]. Samples (50 µL) were obtained and after the appropriate treatment were analyzed by LC-HRMS/MS (ABSciex, Darmstadt, Germany) and the results are shown in Figure 3 (see, also Supplementary Material).

Germany) and the results are shown in Figure 3 (see, also Supplementary Material). An approximately 85% increase on the metabolic stability was observed at 15 min for both compounds; the parent compound remaining was increased from 25% to 45–46%. At 30 min, the effect is smaller, but still significant (70% enhancement, from 17% to 29%). Thus, these methyl substitutions at the left or the right of the oxoester functionality show that when steric hindrance An approximately 85% increase on the metabolic stability was observed at 15 min for both compounds; the parent compound remaining was increased from 25% to 45–46%. At 30 min, the effect is smaller, but still significant (70% enhancement, from 17% to 29%). Thus, these methyl substitutions at the left or the right of the oxoester functionality show that when steric hindrance around the oxoester functionality is increased, the stability of the oxoester in the plasma is increased An approximately 85% increase on the metabolic stability was observed at 15 min for both compounds; the parent compound remaining was increased from 25% to 45–46%. At 30 min, the effect is smaller, but still significant (70% enhancement, from 17% to 29%). Thus, these methyl substitutions at the left or the right of the oxoester functionality show that when steric hindrance around the oxoester functionality is increased, the stability of the oxoester in the plasma is increased as well.

around the oxoester functionality is increased, the stability of the oxoester in the plasma is increased

resultant plots.

resultant plots.

*Biomolecules* **2020**, *10*, x 9 of 12

*Biomolecules* **2020**, *10*, x 9 of 12

**Figure 3.** Plasma stability of GK587 and GK639 in comparison with GK452. **Figure 3.** Plasma stability of GK587 and GK639 in comparison with GK452. **Figure 3.** Plasma stability of GK587 and GK639 in comparison with GK452.

#### *3.3. In Vitro Inhibitory Potency and Selectivity of Compounds GK587 and GK639 3.3. In Vitro Inhibitory Potency and Selectivity of Compounds GK587 and GK639 3.3. In Vitro Inhibitory Potency and Selectivity of Compounds GK587 and GK639*

Both of the synthesized methyl substituted 2-oxoesters were tested for their in vitro inhibitory activity on recombinant human GIVA cPLA2 using a LC/MS lipidomics based mixed micelle assay [25]. In addition, their selectivity over the intracellular human calcium-independent PLA2 (GVIA iPLA2) as well one secreted PLA2 (GV sPLA2) was also studied using similar group-specific mixed micelle assays. The inhibition results presented in Table 1 are expressed as both percent inhibition or as *X*I(50) values. First, the percent of inhibition for each PLA2 enzyme at 0.091 mol fraction of each inhibitor was determined. Then, the *X*I(50) values were measured for compounds that displayed greater than 95% inhibition of GIVA cPLA2. The *X*I(50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme activity by 50%. The dose-response inhibition Both of the synthesized methyl substituted 2-oxoesters were tested for their in vitro inhibitory activity on recombinant human GIVA cPLA2 using a LC/MS lipidomics based mixed micelle assay [25]. In addition, their selectivity over the intracellular human calcium-independent PLA2 (GVIA iPLA2) as well one secreted PLA2 (GV sPLA2) was also studied using similar group-specific mixed micelle assays. The inhibition results presented in Table 1 are expressed as both percent inhibition or as *X*I (50) values. First, the percent of inhibition for each PLA2 enzyme at 0.091 mol fraction of each inhibitor was determined. Then, the *X*I (50) values were measured for compounds that displayed greater than 95% inhibition of GIVA cPLA2. The *X*I (50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme activity by 50%. The dose-response inhibition curves for GIVA cPLA2 inhibitors GK587 and GK639 are shown in Figure 4. Both of the synthesized methyl substituted 2-oxoesters were tested for their in vitro inhibitory activity on recombinant human GIVA cPLA2 using a LC/MS lipidomics based mixed micelle assay [25]. In addition, their selectivity over the intracellular human calcium-independent PLA2 (GVIA iPLA2) as well one secreted PLA2 (GV sPLA2) was also studied using similar group-specific mixed micelle assays. The inhibition results presented in Table 1 are expressed as both percent inhibition or as *X*I(50) values. First, the percent of inhibition for each PLA2 enzyme at 0.091 mol fraction of each inhibitor was determined. Then, the *X*I(50) values were measured for compounds that displayed greater than 95% inhibition of GIVA cPLA2. The *X*I(50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme activity by 50%. The dose-response inhibition

curves for GIVA cPLA2 inhibitors GK587 and GK639 are shown in Figure 4. **Figure 4.** Dose-response inhibition curves for GIVA cPLA2 inhibitors GK587 (**A**) and GK639 (**B**). The curves were generated using GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego CA, USA) with a nonlinear regression targeted at symmetrical sigmoidal curves based on plots of % inhibition vs. log(inhibitor mole fraction). The reported *X*I(50) values were calculated from the curves for GIVA cPLA2 inhibitors GK587 and GK639 are shown in Figure 4. **Figure 4.** Dose-response inhibition curves for GIVA cPLA2 inhibitors GK587 (**A**) and GK639 (**B**). The curves were generated using GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego CA, USA) with a nonlinear regression targeted at symmetrical sigmoidal curves based on plots of % inhibition vs. log(inhibitor mole fraction). The reported *X*I(50) values were calculated from the **Figure 4.** Dose-response inhibition curves for GIVA cPLA2 inhibitors GK587 (**A**) and GK639 (**B**). The curves were generated using GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego CA, USA) with a nonlinear regression targeted at symmetrical sigmoidal curves based on plots of % inhibition vs. log(inhibitor mole fraction). The reported *X*I (50) values were calculated from the resultant plots.

**Compound Structure**

Both compounds GK587 and GK639 were found to inhibit GIVA cPLA2 with *X*I (50) values of 0.00036 and 0.0012, respectively (Table 1). It is apparent that the introduction of a methyl group resulted in reduction of the inhibitory potency, when compared with the parent inhibitor GK452, which exhibits a *X*I (50) value of 0.000078 [23]. However, GK587 was approximately five times less potent than GK452, while GK639 was approximately fifteen times less potent. These findings indicate that the methyl substitution at either position leads to reduced inhibitory activity, however the substitution on the carbon carrying the ester oxygen causes more potent suppression of the potency. That means the methyl substitution on the α-carbon atom to the oxoester functionality is preferable. Both compounds GK587 and GK639 are selective inhibitors of GIVA cPLA2, because both of them did not inhibit GVIA iPLA2 and GV sPLA2 (Table 1). Both compounds GK587 and GK639 were found to inhibit GIVA cPLA2 with *X*I(50) values of 0.00036 and 0.0012, respectively (Table 1). It is apparent that the introduction of a methyl group resulted in reduction of the inhibitory potency, when compared with the parent inhibitor GK452, which exhibits a *X*I(50) value of 0.000078 [23]. However, GK587 was approximately five times less potent than GK452, while GK639 was approximately fifteen times less potent. These findings indicate that the methyl substitution at either position leads to reduced inhibitory activity, however the substitution on the carbon carrying the ester oxygen causes more potent suppression of the potency. That means the methyl substitution on the α-carbon atom to the oxoester functionality is preferable. Both compounds GK587 and GK639 are selective inhibitors of GIVA cPLA2, because both of them did not inhibit GVIA iPLA2 and GV sPLA2 (Table 1). The results shown below lead to the conclusion that the methyl substitution on the α-carbon atom to the oxoester functionality is a successful strategy to increase the plasma stability of the 0.00036 and 0.0012, respectively (Table 1). It is apparent that the introduction of a methyl group resulted in reduction of the inhibitory potency, when compared with the parent inhibitor GK452, which exhibits a *X*I(50) value of 0.000078 [23]. However, GK587 was approximately five times less potent than GK452, while GK639 was approximately fifteen times less potent. These findings indicate that the methyl substitution at either position leads to reduced inhibitory activity, however the substitution on the carbon carrying the ester oxygen causes more potent suppression of the potency. That means the methyl substitution on the α-carbon atom to the oxoester functionality is preferable. Both compounds GK587 and GK639 are selective inhibitors of GIVA cPLA2, because both of them did not inhibit GVIA iPLA2 and GV sPLA2 (Table 1). The results shown below lead to the conclusion that the methyl substitution on the α-carbon atom to the oxoester functionality is a successful strategy to increase the plasma stability of the oxoester inhibitors, ensuring that the inhibitor retains considerable inhibitory potency. which exhibits a *X*I(50) value of 0.000078 [23]. However, GK587 was approximately five times less potent than GK452, while GK639 was approximately fifteen times less potent. These findings indicate that the methyl substitution at either position leads to reduced inhibitory activity, however the substitution on the carbon carrying the ester oxygen causes more potent suppression of the potency. That means the methyl substitution on the α-carbon atom to the oxoester functionality is preferable. Both compounds GK587 and GK639 are selective inhibitors of GIVA cPLA2, because both of them did not inhibit GVIA iPLA2 and GV sPLA2 (Table 1). The results shown below lead to the conclusion that the methyl substitution on the α-carbon atom to the oxoester functionality is a successful strategy to increase the plasma stability of the oxoester inhibitors, ensuring that the inhibitor retains considerable inhibitory potency.

*Biomolecules* **2020**, *10*, x 10 of 12

0.00036 and 0.0012, respectively (Table 1). It is apparent that the introduction of a methyl group resulted in reduction of the inhibitory potency, when compared with the parent inhibitor GK452,

*Biomolecules* **2020**, *10*, x 10 of 12

Both compounds GK587 and GK639 were found to inhibit GIVA cPLA2 with *X*I(50) values of

The results shown below lead to the conclusion that the methyl substitution on the α-carbon atom to the oxoester functionality is a successful strategy to increase the plasma stability of the oxoester inhibitors, ensuring that the inhibitor retains considerable inhibitory potency. oxoester inhibitors, ensuring that the inhibitor retains considerable inhibitory potency. **Table 1.** In vitro Potency and Selectivity of Methyl Substituted 2-Oxoesters. **Table 1.** In vitro Potency and Selectivity of Methyl Substituted 2-Oxoesters. **GIVA cPLA2 GIVA iPLA2 GIVA Table 1.** In vitro Potency and Selectivity of Methyl Substituted 2-Oxoesters. **GIVA cPLA2 GIVA iPLA2 GIVA sPLA2**

**Table 1.** In vitro Potency and Selectivity of Methyl Substituted 2-Oxoesters. **GIVA cPLA2 GIVA iPLA2 Compound Structure % % Inhibitio na** *X***I(50) Inhibitiona**

**%**

**GIVA sPLA2**

**%**

**sPLA2**

**% Inhibitiona**

**%**

**GK639** >95% 0.0012 ± 0.00008 N.D.c N.D.c a % Inhibition at 0.091 mole fraction of each inhibitor; b data taken from ref. [23]. c N.D. signifies a % Inhibition at 0.091 mole fraction of each inhibitor; b data taken from ref. [23]. c N.D. signifies a % Inhibition at 0.091 mole fraction of each inhibitor; b data taken from ref. [23]. c N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).

compounds with less than 25% inhibition (or no detectable inhibition).

a % Inhibition at 0.091 mole fraction of each inhibitor; b data taken from ref. [23]. c N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition). Synthetic GIVA cPLA2 inhibitors are potential novel anti-inflammatory agents. For example, inhibitor GK470, previously developed by our group, was found to suppress the release of arachidonic acid in vitro and to exhibit an anti-inflammatory effect comparable to the reference drug methotrexate, in a prophylactic collagen-induced arthritis model, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel [20]. In addition, synthetic inhibitors may help in clarifying the biological role of GIVA cPLA2 and the inter-connection with compounds with less than 25% inhibition (or no detectable inhibition). Synthetic GIVA cPLA2 inhibitors are potential novel anti-inflammatory agents. For example, inhibitor GK470, previously developed by our group, was found to suppress the release of arachidonic acid in vitro and to exhibit an anti-inflammatory effect comparable to the reference drug methotrexate, in a prophylactic collagen-induced arthritis model, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel [20]. In addition, synthetic inhibitors may help in clarifying the biological role of GIVA cPLA2 and the inter-connection with other enzymes. As an example, the involvement of both PLA2 and phospholipase D (PLD) in the signaling through phosphatidic acid seems important for cancer cell survival [30,31]. Synthetic GIVA cPLA2 inhibitors are potential novel anti-inflammatory agents. For example, inhibitor GK470, previously developed by our group, was found to suppress the release of arachidonic acid in vitro and to exhibit an anti-inflammatory effect comparable to the reference drug methotrexate, in a prophylactic collagen-induced arthritis model, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel [20]. In addition, synthetic inhibitors may help in clarifying the biological role of GIVA cPLA2 and the inter-connection with other enzymes. As an example, the involvement of both PLA2 and phospholipase D (PLD) in the signaling through phosphatidic acid seems important for cancer cell survival [30,31]. Synthetic GIVA cPLA2 inhibitors are potential novel anti-inflammatory agents. For example, inhibitor GK470, previously developed by our group, was found to suppress the release of arachidonic acid in vitro and to exhibit an anti-inflammatory effect comparable to the reference drug methotrexate, in a prophylactic collagen-induced arthritis model, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel [20]. In addition, synthetic inhibitors may help in clarifying the biological role of GIVA cPLA2 and the inter-connection with other enzymes. As an example, the involvement of both PLA2 and phospholipase D (PLD) in the signaling through phosphatidic acid seems important for cancer cell survival [30,31].

other enzymes. As an example, the involvement of both PLA2 and phospholipase D (PLD) in the

#### signaling through phosphatidic acid seems important for cancer cell survival [30,31]. **4. Conclusions 4. Conclusions 4. Conclusions**

**4. Conclusions**  In conclusion, in the present work we designed and synthesized two new 2-oxoester compounds in an effort to increase their metabolic stability. Determining their in vitro plasma stability and their in vitro inhibitory activity on GIVA cPLA2, led us to conclude that inhibitor GK587, in which a methyl group was introduced on the α-carbon atom to the oxoester functionality, exhibits increased metabolic stability retaining at the same time considerable inhibitory potency. In conclusion, in the present work we designed and synthesized two new 2-oxoester compounds in an effort to increase their metabolic stability. Determining their in vitro plasma stability and their in vitro inhibitory activity on GIVA cPLA2, led us to conclude that inhibitor GK587, in which a methyl group was introduced on the α-carbon atom to the oxoester functionality, exhibits increased metabolic stability retaining at the same time considerable inhibitory potency. In conclusion, in the present work we designed and synthesized two new 2-oxoester compounds in an effort to increase their metabolic stability. Determining their in vitro plasma stability and their in vitro inhibitory activity on GIVA cPLA2, led us to conclude that inhibitor GK587, in which a methyl group was introduced on the α-carbon atom to the oxoester functionality, exhibits increased metabolic stability retaining at the same time considerable inhibitory potency. In conclusion, in the present work we designed and synthesized two new 2-oxoester compounds in an effort to increase their metabolic stability. Determining theirin vitro plasma stability and theirin vitro inhibitory activity on GIVA cPLA2, led us to conclude that inhibitor GK587, in which a methyl group was introduced on the α-carbon atom to the oxoester functionality, exhibits increased metabolic stability retaining at the same time considerable inhibitory potency. Thus, including an α-methyl substitution is a promising way of improving the pharmacological properties of 2-oxoester inhibitors and suggests that further chemical modification of this inhibitor class has the potential for pharmaceutical development of potent, metabolically stable, and selective inhibitors of phospholipase A2.

**Supplementary Materials:** HRMS and plasma stability studies as well as copies of the 1H and 13C NMR spectra are available online at http://www.mdpi.com/2218-273X/10/3/491/s1.

**Author Contributions:** Synthesis and writing—original draft preparation, G.S.K.; plasma stability and LC-HRMS studies, M.G.K.; inhibitory activity studies, D.H. and V.D.M.; supervision and writing—review and editing, G.K. and E.A.D. All authors have read and agreed to the published version of the manuscript

**Funding:** This research was carried out within the framework of a Stavros Niarchos Foundation grant to the National and Kapodistrian University of Athens (GK) and NIH grant RO1 GM20501 to the University of California, San Diego (EAD).

**Acknowledgments:** G.S.K. would like to thank Stavros Niarchos Foundation (SNF) for a scholarship.

**Conflicts of Interest:** The authors have declared no conflict of interest.

## **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **cPLA2**α **Enzyme Inhibition Attenuates Inflammation and Keratinocyte Proliferation**

**Felicity J. Ashcroft 1 , Nur Mahammad 1 , Helene Midtun Flatekvål 1 , Astrid J. Feuerherm 1,2 and Berit Johansen 1,\***

Received: 7 September 2020; Accepted: 29 September 2020; Published: 2 October 2020

**Abstract:** As a regulator of cellular inflammation and proliferation, cytosolic phospholipase A2 α (cPLA2α) is a promising therapeutic target for psoriasis; indeed, the cPLA2α inhibitor AVX001 has shown efficacy against plaque psoriasis in a phase I/IIa clinical trial. To improve our understanding of the anti-psoriatic properties of AVX001, we sought to determine how the compound modulates inflammation and keratinocyte hyperproliferation, key characteristics of the psoriatic epidermis. We measured eicosanoid release from human peripheral blood mononuclear cells (PBMC) and immortalized keratinocytes (HaCaT) and studied proliferation in HaCaT grown as monolayers and stratified cultures. We demonstrated that inhibition of cPLA2α using AVX001 produced a balanced reduction of prostaglandins and leukotrienes; significantly limited prostaglandin E2 (PGE2) release from both PBMC and HaCaT in response to pro-inflammatory stimuli; attenuated growth factor-induced arachidonic acid and PGE2 release from HaCaT; and inhibited keratinocyte proliferation in the absence and presence of exogenous growth factors, as well as in stratified cultures. These data suggest that the anti-psoriatic properties of AVX001 could result from a combination of anti-inflammatory and anti-proliferative effects, probably due to reduced local eicosanoid availability.

**Keywords:** cPLA2α; psoriasis; proliferation; anti-inflammatory

#### **1. Introduction**

The phospholipase A2 (PLA2) superfamily of enzymes cleave phospholipids at the sn-2 position to release free fatty acids and lysophospholipids. These are the precursors to a multitude of lipid signaling molecules including the eicosanoids, which are metabolites of arachidonic acid (AA) and have important roles in inflammation and inflammatory diseases. Cytosolic phospholipase A2 α (cPLA2α) is the only PLA2 enzyme with high specificity for phospholipids carrying AA at the sn-2 position, placing it as an important upstream regulator of eicosanoid production [1]. When activated by extracellular stimuli, cPLA2α undergoes Ca++-dependent translocation from the cytoplasm to intracellular membranes and becomes predominantly localized to the peri-nuclear region of the cell [2–4]. This is where metabolism of AA by the cyclo-oxygenase (COX) and lipo-oxygenase (LOX) pathways typically occurs, producing prostaglandins and thromboxane A2 (TxA2), or leukotrienes, hydroxyeicosatetraenoic acids (HETEs), and hydroperoxyeicosatetraenoic acids (HPETEs), respectively. The importance of cPLA2α for stimulus-induced eicosanoid production and the pathogenesis of inflammation has been demonstrated by gene silencing both in vitro [5,6] and in animal models [7–11], and from the use of specific inhibitors of cPLA2α in preclinical models of inflammatory diseases, as was recently reviewed by Nikolaou et al. [12]. Examples include the use of the indole-derivative

ZPL-5212372 in asthma and atopic dermatitis [13], the pyrrolidine-based compound RSC-3388 in a *Streptococcus pneumonia* infection model [14], and the ω-3 polyunsaturated fatty acid (PUFA) derivatives AVX001 and AVX002 in collagen-induced arthritis [15].

Plaque psoriasis (psoriasis vulgaris) is a disease with a chronic inflammatory phenotype that drives the hyperproliferation and aberrant differentiation of the epidermis [16]. Chronic inflammation in psoriasis is associated with higher expression of PLA2 enzymes [17–19] and increased levels of eicosanoids [20–23]. Evidence for the involvement of eicosanoids in psoriasis is supported by mouse models of the disease—the leukotriene B4 (LTB4) receptor 1 and TxA2 receptor have critical roles in imiquimod-induced skin inflammation [24–26], and prostaglandin E2 (PGE2) acting at prostaglandin receptors EP2 and EP4 is important for Th17-dependent inflammation in interleukin 23 (IL-23)-induced psoriasis [27]. Suppression of eicosanoid production is therefore an interesting prospect for treating psoriasis.

Non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit AA metabolism via the COX pathway are commonly used for their analgesic, anti-inflammatory, and antithrombotic actions; however, their use is associated with many adverse gastrointestinal and cardiovascular effects (as reviewed in [28]) and they can induce or exacerbate psoriasis. The latter effect at least is postulated to be attributable to a skewed eicosanoid profile and the accumulation of leukotrienes (reviewed in [29,30]). Thus, it has been hypothesized that creating a more balanced suppression of eicosanoids using either dual COX-LOX inhibitors or by suppression of AA production using PLA2 inhibitors (reviewed in [12,31]) would provide a better and safer therapeutic option.

The cPLA2α inhibitor AVX001 is a ω-3 PUFA-derivative developed by Avexxin (now Coegin Pharma) that was demonstrated to be highly selective and to inhibit the in vitro activity of cPLA2α with an IC50 of 120nM, being more potent than either docosahexaenoic acid (DHA) or the ω-6 PUFA derivative arachidonyl trifluoromethyl ketone (AACOCF3, ATK) [15,32]. A topical application of AVX001 was trialed in a randomized, double-blind, placebo-controlled, dose-escalation first-in-man study to assess its safety and efficacy in patients with mild to moderate plaque psoriasis [33]. AVX001 showed significant efficacy and was well tolerated up to the maximum dose tested of 5%, supporting the targeting of cPLA2α as a safe therapeutic strategy. The specificity and potency of cPLA2α inhibition by AVX001 has been demonstrated [15,32], however, its mode of action in psoriasis remains to be determined. Given the documented role of cPLA2α in mediating inflammatory signals in monocytes and keratinocytes [34–37] and the more recent interest in cPLA2α as a driver of cellular proliferation [38], we sought to study potential modes of action of AVX001 in psoriatic skin by investigating its effects on inflammation and proliferation using human peripheral blood mononuclear cells (PBMC) and keratinocytes.

#### **2. Materials and Methods**

#### *2.1. Materials*

Cell culture media and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, US) unless stated otherwise. A23178, naproxen, celecoxib calcipotriol hydrate, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. Nordihydroguaiaretic acid (NDGA) was from Cayman chemicals (Ann Arbor, MI, USA) Recombinant human epidermal growth factor (EGF) and tumour necrosis factor (TNF)-α were from R&D systems (Abdingdon, UK). The fluoroketone AVX001 was synthesized and characterized according to Holmeide and Skattebol [39], and provided by Dr. Inger Reidun Aukrust and Dr. Marcel Sandberg (Synthetica AS, Oslo, Norway). AVX001 was stored at −80 ◦C as a 20 mM stock solution in dimethyl sulphoxide (DMSO) under argon gas to minimize oxidation.

#### *2.2. PBMC Isolation and Treatment*

Blood was recruited from healthy donors at St. Olavs Hospital HF, the Bloodbank (project approved by Regional Ethical Committee of Mid-Norway; #2016/553). Peripheral blood mononuclear cells (PBMC) were isolated using SepMate separation tubes with LymphoPrep density gradient medium from STEMCELL Technologies (Cambridge, UK), according to the manufacturer's recommendations. For experiments, 1 x 106 cells per well were plated in 1 mL Roswell Park Memorial Insitute (RPMI) medium supplemented with 5% fetal bovine serum (FBS), 0.3 mg/mL glutamine, and 0.1 mg/mL gentamicin. Inhibitors were added 2 h prior to the addition of the Ca++ ionophore A23178 (30 µM, 15 min) to activate cPLA2α or lipopolysaccharide (LPS) (10 ng/mL, 72 h) as a potent inducer of inflammation. Following treatment, the cell suspensions were centrifuged to isolate the supernatant from the cell fraction. Samples were stored at −80 ◦C until analysis.

#### *2.3. Enzyme-Linked Immunoassay Detection of Eicosanoids*

Cell supernatant samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for PGE2 (Cayman #514435), LTB4 (Cayman #10009292), TxB2 (Cayman #501020), or 12S-HETE (Enzo Lifesciences #ADI-900-050) according to the manufacturers' protocols. Cell supernatants were assayed at dilutions of 1:100 for PGE2, except supernatants from non-LPS-treated PBMC that were assayed undiluted in all assays. Supernatants were hybridized overnight, and the enzymatic conversion of the substrate was read at OD420 nm. Data were processed using a 4-parameter logistic fit model.

#### *2.4. Culture of HaCaT Keratinocytes*

#### 2.4.1. Maintenance

The spontaneously immortalized skin keratinocyte cell line HaCaT [40] was kindly provided by Prof. N. Fusenig (Heidelberg, Deutsches Krebsforschungszentrum, Germany). These cells are commonly used to study proliferative and inflammatory responses in psoriasis research [41–46], as they express epidermal growth factor receptor (EGFR) and can proliferate both independently of, as well as in response to, stimulation with growth factors [47]. HaCaT were maintained in Dulbecco's modified Eagle Medium (DMEM) supplemented with 5% (*v*/*v*) FBS, 0.3 mg/mL glutamine, and 0.1 mg/mL gentamicin (DMEM-5) at 37 ◦C with 5% CO2 in a humidified atmosphere at sub-confluency to prevent differentiation. Treatments were carried out in DMEM supplemented with 0.5% (*v*/*v*) FBS and 0.3 mg/mL glutamine (DMEM-0.5)

#### 2.4.2. Eicosanoid Release

For analysis of eicosanoid release, we plated HaCaT in 12-well plates at 5 × 104 cells per well in DMEM-5 and cultured them for 3 days until reaching approximately 50% confluency, when the media was replaced with DMEM-0.5. The following day, the cells were stimulated with tumour necrosis factor (TNF)-α (30 ng/mL, 72 h), EGF (30 ng/mL, 24 h), or calcipotriol (10 nM, 72 h).

#### *2.5. [3H]-Arachidonic Acid Release Assay*

At 2 days post-confluency, we labelled HaCaT for 18 h with 3H-AA (0.4 µCi/mL) in DMEM-0.5. After labelling, the cells were washed twice with phosphate-buffered saline (PBS) containing fatty acid-free bovine serum albumin (BSA) (2 mg/mL) in order to remove unincorporated radioactivity. After stimulation (EGF 100 ng/mL, 60 min), the supernatants were cleared of detached cells by centrifugation (13,000 rpm, 10 min). The release of 3H-AA from the cells was assessed by liquid scintillation counting in a LS 6500 Multi-Purpose Scintillation Counter (Beckman Coulter, Brea, CA, USA). Adherent cells were dissolved in 1M NaOH in order to determine incorporated 3H-AA in the cells by liquid scintillation counting. The results are given as released 3H-AA in the supernatants relative to total 3H-AA incorporated into the cells.

#### *2.6. Resazurin Assay*

HaCaT were seeded in 96-well plates in DMEM-5 at a density of 3000 cells per well. Following 72 h of cultivation, when cells reached a density of approximately 50%, we replaced the medium with DMEM-0.5. The following day, the cells were treated with AVX001, in a series of eight wells per treatment, for 24h. Resazurin (RnD systems, Abingdon, United Kingdom) was added according to the manufacturer's instructions and left to incubate for 2h at 37 ◦C with 5% CO2 in a humidified atmosphere. Fluorescence was read at 544 nm excitation and 590 nm emission wavelengths using the Cytation 5 cell imaging multimode reader (Biotek Instruments, Winooski, VT, USA).

#### *2.7. High Throughput Microscopy Assay for Population Analysis of Cell Cycle and Apoptosis*

Cells were seeded in Greiner Bio-one CELLSTAR 96-well flat clear flat-bottomed plates (BioNordika, Oslo, Norway) in DMEM-5 at a density of 3000 cells per well. After 72 h, when cells reached a density of approximately 50%, we replaced the medium with DMEM-0.5. The following day, cells were treated with vehicle, AVX001, or etoposide (10 µM) in DMEM-0.5 for 24 h. We then followed the manufacturer's guidelines for the Click-iT 5-ethyl-20 -deoxyuridine (EdU) Alexa Fluor 594 imaging kit (ThermoFisher Scientific, Waltham, MA, USA) using a final concentration of 10 µM EdU per well incubated for a further 2 h at 37 ◦C, 5% CO2. Following incubation with EdU, we removed the media and replaced it with the CellEvent Caspase 3/7 Green detection reagent (ThermoFisher Scientific) prepared at 2 µM in Dulbecco's (D)-PBS +5% FBS. The cells were incubated for a further 45 min, then the reagent was removed, and the cells were immediately fixed using 4% formaldehyde in D-PBS for 20 min on ice. Permeabilization was carried out using 0.1% Triton-X 100 in D-PBS and the Click-iT reaction was performed according to the manufacturer's guidelines using the Alexa-594 picoyl azide to label incorporated EdU. Finally, the cells were counterstained by incubation with 1 µg/mL 4 0 ,6-diamidino-2-phenylindole (DAPI) (ThermoFisher Scientific) in D-PBS for 5 min. DAPI solution was removed and replaced with D-PBS for imaging. Plates were stored in the dark at 4 ◦C. All steps were performed at room temperature unless otherwise stated. Automated imaging was carried out on the Cytation5 cell imaging multimode reader (Biotek Instruments) at 4× magnification using DAPI, TexasRed, and GFP filter sets to image the DAPI, Alexa-594, and CellEvent Green signals, respectively. Four images were taken per well and 3 wells per treatment were used for the analysis.

Image analysis was performed in the freeware CellProfiler version 3.1.9 [48]. Firstly, nuclei were segmented from DAPI images using an Ostu 2-class thresholding approach, and were then counted. In further analyses, filters were employed to remove images with fewer than 50 cells. For cell cycle analysis, the total DAPI intensity and total EdU staining were then measured per nuclei from 12 images per treatment group and the freeware Flowing version 2.5.1 (Perttu Terho, Turku Centre for Biotechnology) was used to identify cells in G1, G2, and S-phases of the cell cycle, with gating based on log10 total EdU intensity vs. total DNA intensity. Apoptotic cells were identified on the basis of robust-background thresholding of the CellEvent Green signal and reported as a percentage of the total number of cells per image. Four images were taken per well and data were based on 3 wells per treatment group. To calculate the proliferative index, we segmented EdU-positive cells using an Ostu 2-class thresholding approach and reported them as a proportion of the total number of cells per image. Four images were taken per well and data were based on 3 wells per treatment group.

#### *2.8. RNA Extraction and Real-Time Quantitative PCR*

Cells were seeded in 6-well plates in DMEM-5. Following 72 h of cultivation, when cells reached a density of approximately 50%, we replaced the medium with DMEM-0.5. The following day, the cells were preincubated with AVX001 for 2 h prior to stimulation with EGF (30 ng/mL, 4 h). Total RNA was extracted with Total RNA kit I from Omega BIO-TEK (Norcross, GA, USA) according to the manufacturer0 s protocol. The amount and purity of the RNA samples were quantified using a Nanodrop One/OneC Microvolume UV–VIS Spectrophotometer (ND-ONE-W) from ThermoFisher Scientific. RNA samples with absorbance (A) A260/A230 between 1.8 and 2.1 and A260/280 between 2.0 and 2.2 were accepted. Reverse transcription was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) with 1 µg of RNA per sample, according to the manufacturer's protocol. Real-time PCR analysis was performed using the LightCycler 480 SYBR Green I Master MIX and LightCycler 96 instrument from Roche (Basel, Switzerland), according to the manufacturer's protocol.

#### *2.9. 3D Culture of HaCaT Keratinocytes*

3D stratified HaCaT cultures were grown in Nunc cell culture inserts (0.4 µm pore size) using the 24-well carrier plates system (Thermo Fisher Scientific #141002) The culture inserts were coated using the Coating Matrix kit (Thermofisher Scientific #R-011-K) according to the manufacturer's protocol. HaCaT were plated at a density of 0.3 × 105 cells per insert in 0.5 mL DMEM-5 and incubated for 24 h before being lifted to the air–liquid interface. The media in the lower chamber was replaced with DMEM-5 (without antibiotics) + 1 ng/mL EGF, and 5 µg/mL L-ascorbic acid in the absence or presence of AVX001 (5 µM). The media in the lower chambers and treatments were changed every 3rd day for 12 days.

The cultures were fixed in 4% paraformaldehyde (PFA) overnight, before processing for paraffin embedding. Briefly, the membranes were removed from the inserts and prepared in Tissue Clear (Sakura, Osaka, Japan) for paraffin wax embedding using the Excelsior AS Tissue processor (ThermoFisher Scientific). Paraffin embedded sections (4 µm) were cut onto SuperFrost Plus slides (ThermoFisher scientific), dried over night at 37 ◦C, and then baked for 60 min at 60 ◦C. The sections were dewaxed in Tissue Clear and rehydrated through graded alcohols to water in an automatic slide stainer (Tissue-Tek Prisma, Sakura). Next, the sections were pretreated in Target Retrieval Solution, High pH (Dako, Glostrup, Denmark, K8004) in PT Link (Dako) for 20 min at 97 ◦C to facilitate antigen retrieval. The staining was performed according to the manufacturer's procedure with EnVision G|2 Doublestain System Rabbit/Mouse (DAB+/Permanent Red) kit (Dako/Agilent K5361) on the Dako Autostainer. Following their soaking in wash buffer, we quenched endogenous peroxidase and alkaline phosphatase activity with Dual Endogenous Enzyme Block (Dako). Sections were then rinsed in wash buffer and incubated with primary antibody against Ki67 (MIB1 (Dako M7240) diluted 1:300) for 40 min. The slides were rinsed before incubating in horseradish peroxidase (HRP) - polymer and 3,30 -Diaminobenzidine (DAB) to develop the stain. After a double stain block, the sections were incubated in antibody against cytokeratin 10 (Invitrogen #MA5-13705 diluted 1:100) for 60 min. After incubation in the mouse/rabbit linker, the sections were incubated in AP- polymer and the corresponding red substrate buffer with washing between each step. Tris-buffered saline (TBS; Dako K8007) was used throughout for the washing steps. The slides were lightly counterstained with hematoxylin, completely dried, and coverslipped. Appropriate negative controls were performed; both mouse monoclonal isotype control (Biolegend, San Diego, CA, USA) and omitting the primary antibody (negative method control).

#### *2.10. Statistical Analysis*

Statistical analysis was carried out in GraphPad Prism Software, version 7, using one-way ANOVA with Dunnet's post-analysis. For normalized data, we used the Kruskal–Wallis test with Dunn's post-analysis.

#### **3. Results**

#### *3.1. Inhibition of cPLA2*α *Using AVX001 Resulted in a Balanced Reduction of Eicosanoids*

Several eicosanoids including PGE2, prostaglandin F2 (PGF2), LTB4, and 12S-HETE are known to be elevated in psoriatic lesions [20–23,49], and their direct roles in disease progression are supported by animal models of inflammatory skin disease [24,31,50,51]. Hence, it has been proposed that generating a balanced reduction in overall eicosanoid production is a promising therapeutic strategy [12,31]. To test whether the cPLA2α inhibitor AVX001 can normalize a broader range of eicosanoids than known COXand LOX inhibitors, we treated PBMC with AVX001, the non-specific lipo-oxygenase (LOX) inhibitor nordihydroguaiaretic acid (NDGA), the cyclooxygenase (COX)-2 selective inhibitor celecoxib, or the

dual COX1/COX2 inhibitor naproxen, and stimulated the mixture with the Ca++ ionophore A23178. The levels of PGE2, TxB2, LTB4, and 12S-HETE were assayed by ELISA and inhibition was calculated as a percentage of the A23178 stimulation. The maximum inhibition and concentration at which this was reached is presented in Table 1, and the absolute eicosanoid levels are available in Appendix A (Figure A1). We observed that AVX001 treatment dose-dependently inhibited the release of PGE2, TxB2, and LTB4. AVX001 also reduced 12S-HETE release at the maximum dose tested (10 µM), although it did not reach significance (*p* = 0.12, *n* = 3). As expected, NDGA dose-dependently inhibited the release of LOX metabolites LTB4 and 12S-HETE, but not COX metabolites PGE2 and TxB2. Celecoxib treatment gave a dose-dependent reduction of PGE2, TxB2, and LTB4 but did not inhibit 12S-HETE. The finding that the COX-2 selective inhibitor celecoxib reduced LTB4 release was surprising, however, it was consistent with the following study that demonstrated inhibition of 5-LOX but not 12- or 15-LOX by celecoxib in human blood [52]. Naproxen treatment, on the other hand, gave dose-dependent inhibition of the COX metabolites PGE2 and TxB2 without affecting LOX metabolites LTB4 or 12S-HETE. AVX001 treatment was therefore shown to result in a broader inhibition of eicosanoid release than the COX or LOX inhibitors tested, having a similar efficacy to reduce both COX and 5-LOX metabolites, and possibly also the 12-LOX metabolite 12S-HETE, albeit at a higher dose.

**Table 1.** Inhibition of eicosanoid release in A23178-stimulated peripheral blood mononuclear cells (PBMC). Eicosanoid levels were measured in the supernatants of PBMC pre-incubated with AVX001, naproxen, celecoxib, or nordihydroguaiaretic acid (NDGA) in the range of 0.5-10 µM and stimulated with A23178 (30 µM, 15 min). The percentage inhibition of the A23178-stimulated release was calculated for each eicosanoid and is shown as the mean ± standard error of the mean (SEM) for *n* = 4 (prostaglandin E2 (PGE2 )), *n* = 4 (leukotriene B4 (LTB4 )), and *n* = 3 (12S-hydroxyeicosatetranoic acid (HETE)) individuals. The inhibitor concentration at which maximal inhibition was reached is given below. Statistical significance was calculated using Kruskal–Wallis tests with Dunn's post-analysis; \* *p* < 0.05, \*\* *p* < 0.01. A graphical presentation of absolute eicosanoid levels is available in Figure A1.

## *3.2. AVX001 Inhibited PGE2 Release in Response to Inflammatory Stimuli*

AVX001 inhibited eicosanoid release from A23187-stimulated PBMC, however, the use of the Ca++ ionophore does not represent a physiological stimulus, and therefore it was important to confirm the effects using biologically relevant stimuli. For these experiments, we measured the PGE2 release. This was selected because PGE2 is one of the main eicosanoids produced in the skin, being released by epidermal keratinocytes, dermal fibroblasts, and immune cells. It has proinflammatory and immuno-modulatory properties and promotes keratinocyte proliferation (reviewed in [53]). We measured PGE2 levels in response to inflammatory stimuli and epidermal growth factor (EGF) and investigated the role of cPLA2α in these responses.

To investigate the role of cPLA2α in the response to pro-inflammatory stimuli, we preincubated human PBMC or HaCaT with AVX001 and stimulated them with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α, respectively. LPS stimulated PGE2 release in PBMC by an average of 91-fold. Pretreatment with AVX001 dose-dependently inhibited LPS-stimulated PGE2 release with an IC50 of

5 µM (Figure 1A). HaCaT released a low but detectable level of PGE2 that was stimulated by TNF-α treatment by an average of 67-fold. AVX001 also dose-dependently inhibited TNF-α-stimulated PGE2 release (Figure 1B). These data support a role for cPLA2α in mediating pro-inflammatory eicosanoid release from immune cells and keratinocytes, suggesting that AVX001 would have anti-inflammatory properties in the skin. *Biomolecules* **2020**, *10*, x 7 of 18

**Figure 1.** Effect of AVX001 on pro-inflammatory eicosanoid production. (**A**) PBMC were preincubated with AVX001 (µM) and stimulated with lipopolysaccharide (LPS) (10 ng/mL, 72 h). (**B**) Immortalized keratinocytes (HaCaT) were pre-incubated with AVX001 (µM) and stimulated with tumor necrosis factor (TNF)-α (10 ng/mL, 72 h). (**C**) HaCaT were pre-incubated with AVX001 (µM) and stimulated with calcipotriol (10 nM, 72 h). (**D**) (**i**) PBMC were treated with calcipotriol (µM) for 72 h, (**ii**) PBMC were pre-treated with AVX001 (µM) and stimulated with calcipotriol (10 nM, 72 h). (**E**) PBMC were pre-incubated with AVX001 (µM) and stimulated with calcipotriol (10 nM, 72 h) or vehicle in the presence of LPS (10 ng/mL). PGE2 levels in supernatants were determined by ELISA and reported as either pg/mL (**A**–**C**), or as the fold change in PGE2 levels over unstimulated (**D**) or LPS-stimulated (**E**) controls. Data are the mean ± SEM of ≥ 4 individuals (PBMC) or 3 replicates (HaCaT). Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis, or, for normalized data, using the Kruskal–Wallis test with Dunn's post-analysis; #*p* < 0.05, ##*p* < 0.01, or ###*p* < 0.005 versus unstimulated control and \**p* < 0.05, \*\**p* < 0.01 versus vehicle-treated control. **Figure 1.** Effect of AVX001 on pro-inflammatory eicosanoid production. (**A**) PBMC were pre-incubated with AVX001 (µM) and stimulated with lipopolysaccharide (LPS) (10 ng/mL, 72 h). (**B**) Immortalized keratinocytes (HaCaT) were pre-incubated with AVX001 (µM) and stimulated with tumor necrosis factor (TNF)-α (10 ng/mL, 72 h). (**C**) HaCaT were pre-incubated with AVX001 (µM) and stimulated with calcipotriol (10 nM, 72 h). (**D**) (**i**) PBMC were treated with calcipotriol (µM) for 72 h, (**ii**) PBMC were pre-treated with AVX001 (µM) and stimulated with calcipotriol (10 nM, 72 h). (**E**) PBMC were pre-incubated with AVX001 (µM) and stimulated with calcipotriol (10 nM, 72 h) or vehicle in the presence of LPS (10 ng/mL). PGE2 levels in supernatants were determined by ELISA and reported as either pg/mL (**A**–**C**), or as the fold change in PGE2 levels over unstimulated (**D**) or LPS-stimulated (**E**) controls. Data are the mean ± SEM of ≥ 4 individuals (PBMC) or 3 replicates (HaCaT). Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis, or, for normalized data, using the Kruskal–Wallis test with Dunn's post-analysis; # *p* < 0.05, ## *p* < 0.01, or ### *p* < 0.005 versus unstimulated control and \* *p* < 0.05, \*\* *p* < 0.01 versus vehicle-treated control.

Skin toxicity is a common side-effect of a variety of drug types including the vitamin D receptor (VDR) agonist calcitriol and its analogue calcipotriol, both of which are used to treat psoriasis [55]. The pro-inflammatory effects of VDR agonists are suggested to involve PGE2 [56,57]. To investigate the role of cPLA2α in VDR agonist-mediated PGE2 release, we treated PBMC and HaCaT with calcipotriol in the absence or presence of AVX001. We observed a small but significant increase in PGE2 in HaCaT exposed to calcipotriol; pre-incubation with AVX001 reduced the stimulatory effect, reaching marginal significance (*p* < 0.1) at the highest dose used (3 μM) (Figure 1C). Exposure to Skin toxicity is a common side-effect of a variety of drug types including the vitamin D receptor (VDR) agonist calcitriol and its analogue calcipotriol, both of which are used to treat psoriasis [54]. The pro-inflammatory effects of VDR agonists are suggested to involve PGE2 [55,56]. To investigate the role of cPLA2α in VDR agonist-mediated PGE2 release, we treated PBMC and HaCaT with calcipotriol in the absence or presence of AVX001. We observed a small but significant increase in PGE2 in HaCaT exposed to calcipotriol; pre-incubation with AVX001 reduced the stimulatory effect, reaching marginal significance (*p* < 0.1) at the highest dose used (3 µM) (Figure 1C). Exposure to calcipotriol also

calcipotriol also stimulated the release of PGE2 from PBMC by approximately twofold; pre-incubation with AVX001 (5 µM) blocked the calcipotriol-induced increase in PGE2 (Figure 1D). Calcipotriol also

high variability between the responses meant that this was not significant overall. In cases where PGE2 levels were increased, AVX001 (5 µM) inhibited the response (Figure 1E). Our data support the hypothesis that adverse skin reactions resulting from calcipotriol treatment could result from increased PGE2 and suggest that AVX001 may be useful to abrogate these reactions by limiting PGE2.

stimulated the release of PGE2 from PBMC by approximately twofold; pre-incubation with AVX001 (5 µM) blocked the calcipotriol-induced increase in PGE2 (Figure 1D). Calcipotriol also augmented the LPS-stimulated release of PGE2 in PBMC from a subset of individuals, although the high variability between the responses meant that this was not significant overall. In cases where PGE2 levels were increased, AVX001 (5 µM) inhibited the response (Figure 1E). Our data support the hypothesis that adverse skin reactions resulting from calcipotriol treatment could result from increased PGE2 and suggest that AVX001 may be useful to abrogate these reactions by limiting PGE2. *Biomolecules* **2020**, *10*, x 8 of 18

#### *3.3. AVX001 Inhibited EGF-Stimulated Release of AA and PGE2. 3.3. AVX001 Inhibited EGF-Stimulated Release of AA and PGE2.*

Epidermal growth factor receptor (EGFR) activation is a well-known mitogenic signal for epidermal keratinocytes [57] and can activate cPLA2α via mitogen-activated protein kinase (MAPK)-dependent phosphorylation [3,58]. To investigate the effect of AVX001 on growth factor-mediated cPLA2α activation and eicosanoid release in keratinocytes, we stimulated HaCaT with EGF in the absence or presence of AVX001 and measured AA release and PGE2 levels. Consistent with activation of cPLA2α, EGF stimulated the release of both AA and PGE2 in HaCaT (Figure 2.) Treatment with AVX001 (≥ 0.3 µM) significantly and dose-dependently inhibited EGF-stimulated AA (Figure 2A) and PGE2 (Figure 2B) release. Epidermal growth factor receptor (EGFR) activation is a well-known mitogenic signal for epidermal keratinocytes [58] and can activate cPLA2α via mitogen-activated protein kinase (MAPK) dependent phosphorylation [3,59]. To investigate the effect of AVX001 on growth factor-mediated cPLA2α activation and eicosanoid release in keratinocytes, we stimulated HaCaT with EGF in the absence or presence of AVX001 and measured AA release and PGE2 levels. Consistent with activation of cPLA2α, EGF stimulated the release of both AA and PGE2 in HaCaT (Figure 2.) Treatment with AVX001 (≥ 0.3 µM) significantly and dose-dependently inhibited EGF-stimulated AA (Figure 2A) and PGE2 (Figure 2B) release.

**Figure 2.** Effect of cPLA2α inhibition on epidermal growth factor (EGF)-dependent arachidonic acid (AA) and PGE2 release. (**A**) AA release was measured by the [3H]-arachidonic acid release assay in HaCaT treated with AVX001 and stimulated with EGF (100 ng/mL, 60 min). Data are the mean ± SD for three replicates from a representative experiment repeated twice. (**B**) PGE2 levels measured in the supernatant of cells preincubated with AVX001 at the indicated concentrations and stimulated with EGF (30 ng/mL, 24 h). Data are mean ± SEM for three replicates. Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis; #*p* < 0.05, ##*p* < 0.01, or ###*p* < 0.005 versus unstimulated control and \**p* < 0.05, \*\**p* < 0.01 versus vehicle-treated control. **Figure 2.** Effect of cPLA2α inhibition on epidermal growth factor (EGF)-dependent arachidonic acid (AA) and PGE2 release. (**A**) AA release was measured by the [3H]-arachidonic acid release assay in HaCaT treated with AVX001 and stimulated with EGF (100 ng/mL, 60 min). Data are the mean ± SD for three replicates from a representative experiment repeated twice. (**B**) PGE2 levels measured in the supernatant of cells preincubated with AVX001 at the indicated concentrations and stimulated with EGF (30 ng/mL, 24 h). Data are mean ± SEM for three replicates. Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis; # *p* < 0.05, ### *p* < 0.005 versus unstimulated control and \* *p* < 0.05, \*\* *p* < 0.01 versus vehicle-treated control.

#### *3.4. AVX001 Inhibited Keratinocyte Proliferation 3.4. AVX001 Inhibited Keratinocyte Proliferation*

We showed that inhibition of cPLA2α activity using AVX001 attenuated both AA and PGE2 release stimulated by the addition of exogenous growth factor in HaCaT. In fibroblasts, this signaling cascade was required for cell cycle progression [38]. We thus hypothesized that the treatment of keratinocytes with AVX001 could inhibit proliferation. To test this, we first treated actively proliferating HaCaT cells with AVX001 for 24 h and measured cell viability using resazurin as an indicator of metabolically active cells. We then quantified cell number, cell cycle distribution, and apoptosis using a high content image-based assay based on the fluorescent labeling and single-cell quantification of DNA, incorporated EdU, and caspase-3/7 activity, as detailed in Section 2.7. AVX001 inhibited HaCaT viability with an IC50 of 8.5 µM (Figure 3A). The reduced viability observed using We showed that inhibition of cPLA2α activity using AVX001 attenuated both AA and PGE2 release stimulated by the addition of exogenous growth factor in HaCaT. In fibroblasts, this signaling cascade was required for cell cycle progression [38]. We thus hypothesized that the treatment of keratinocytes with AVX001 could inhibit proliferation. To test this, we first treated actively proliferating HaCaT cells with AVX001 for 24 h and measured cell viability using resazurin as an indicator of metabolically active cells. We then quantified cell number, cell cycle distribution, and apoptosis using a high content image-based assay based on the fluorescent labeling and single-cell quantification of DNA, incorporated EdU, and caspase-3/7 activity, as detailed in Section 2.7. AVX001 inhibited HaCaT viability with an IC50 of 8.5 µM (Figure 3A). The reduced viability observed using 10 µM AVX001 was

etoposide, a chemotherapy drug known to block cell cycle progression in HaCaT cells [60–62]. As expected, treatment with the etoposide (10 µM) caused a decrease in cell numbers that, in contrast to AVX001, was associated with an accumulation of cells in G2/M, and an increased proportion of

apoptotic cells, consistent with its known effect as a blocker of the G2/M transition [63,64].

10 µM AVX001 was associated with a reduction in the total cell count (Figure 3Bi) and a reduction in

associated with a reduction in the total cell count (Figure 3B) (i) and a reduction in the proportion of the cells in S-phase (Figure 3B) (ii); there was no significant difference in the number of apoptotic cells (Figure 3B) (iii). The data also suggested an accumulation of cells in G1, although this finding was not statistically significant (*p* = 0.06). As a positive control, cells were treated with etoposide, a chemotherapy drug known to block cell cycle progression in HaCaT cells [59–61]. As expected, treatment with the etoposide (10 µM) caused a decrease in cell numbers that, in contrast to AVX001, was associated with an accumulation of cells in G2/M, and an increased proportion of apoptotic cells, consistent with its known effect as a blocker of the G2/M transition [62,63]. *Biomolecules* **2020**, *10*, x 9 of 18

**Figure 3.** AVX001 inhibited proliferation in HaCaT monolayers. (**A**) Cell viability was measured using the resazurin assay in proliferating HaCaT treated with AVX001 for 24 h at the concentrations indicated. Measurements were normalized to the vehicle-treated control, and the mean ± SEM for three replicates is shown. (**B**) Automated microscopy and analysis of fluorescently labeled DNA, incorporated EdU, and caspase-3/7 activity was used to quantify (**i**) cell number, (**ii**) cell cycle distribution, and (**iii**) apoptosis in proliferating HaCaT treated with AVX001 for 24 h at the concentrations indicated. Data are mean ± SEM for ≥4 replicates. (**C**) (**i**) The proportion of cells in Sphase of the cell cycle (proliferation index) was determined by counting the total and EdU-positive nuclei in proliferating HaCaT pre-incubated with vehicle or AVX001 and stimulated with EGF (30ng/mL, 24 h). Data are the mean ± SEM for three replicates from a representative experiment, repeated twice. (**ii**) Representative images showing 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei (DAPI) and fluorescently labelled EdU (EdU) for unstimulated HaCaT (control), and HaCaT stimulated with EGF (30 ng/mL, 24 h) in the absence (+EGF) or presence of 7 µM AVX001 (AVX001+EGF). (**D**) The relative expression of cyclin D1 measured by quantitative PCR in **Figure 3.** AVX001 inhibited proliferation in HaCaT monolayers. (**A**) Cell viability was measured using the resazurin assay in proliferating HaCaT treated with AVX001 for 24 h at the concentrations indicated. Measurements were normalized to the vehicle-treated control, and the mean ± SEM for three replicates is shown. (**B**) Automated microscopy and analysis of fluorescently labeled DNA, incorporated EdU, and caspase-3/7 activity was used to quantify (**i**) cell number, (**ii**) cell cycle distribution, and (**iii**) apoptosis in proliferating HaCaT treated with AVX001 for 24 h at the concentrations indicated. Data are mean ± SEM for ≥4 replicates. (**C**) (**i**) The proportion of cells in S-phase of the cell cycle (proliferation index) was determined by counting the total and EdU-positive nuclei in proliferating HaCaT pre-incubated with vehicle or AVX001 and stimulated with EGF (30 ng/mL, 24 h). Data are the mean ± SEM for three replicates from a representative experiment, repeated twice. (**ii**) Representative images showing 40 ,6-diamidino-2-phenylindole (DAPI)-stained nuclei (DAPI) and fluorescently labelled EdU (EdU) for unstimulated HaCaT (control), and HaCaT stimulated with EGF (30 ng/mL, 24 h) in the absence (+EGF) or presence of 7 µM AVX001 (AVX001+EGF). (**D**) The relative expression of cyclin D1 measured by quantitative PCR in proliferating HaCaT pre-treated with AVX001 and stimulated with EGF (30 ng/mL, 4h). Data were normalized to the unstimulated control (control) and are the mean ± SEM from six replicates. Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis, or, for normalized data, the Kruskal–Wallis test with Dunn's post-analysis; # *p* < 0.05, ## *p* < 0.01, or ### *p* < 0.005 versus unstimulated control and \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.005 versus vehicle-treated control.

Treatment with EGF for 24 h did not significantly impact cell viability (not shown), but rather led to an increase in the proportion of cells in S-phase of the cell cycle (proliferation index), which was inhibited by AVX001 (≥5 μM) (Figure 3C). Progression of the cell cycle from G1 to S-phase in response to growth factors is typically associated with increased levels of cyclin D1 [65]. Following 4 h of EGF treatment, cyclin D1 transcript levels were increased approximately twofold in HaCaT, and

proliferating HaCaT pre-treated with AVX001 and stimulated with EGF (30 ng/mL, 4h). Data were normalized to the unstimulated control (control) and are the mean ± SEM from six replicates. Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis, or, for

Treatment with EGF for 24 h did not significantly impact cell viability (not shown), but rather led to an increase in the proportion of cells in S-phase of the cell cycle (proliferation index), which was inhibited by AVX001 (≥5 µM) (Figure 3C). Progression of the cell cycle from G1 to S-phase in response to growth factors is typically associated with increased levels of cyclin D1 [64]. Following 4 h of EGF treatment, cyclin D1 transcript levels were increased approximately twofold in HaCaT, and this was inhibited by AVX001 (5 µM) (Figure 3D). These findings support cPLA2α as a target for inhibiting growth factor-dependent proliferation by halting cells in G1.

AVX001 inhibited cell proliferation in HaCaT grown in monolayers; however, experiments performed in 3D culture systems are often considered to have more physiological relevance. HaCaT retain the ability to stratify and differentiate in culture [65,66], and this is dependent on the presence of exogenous growth factors [67]. To test whether AVX001 is an effective inhibitor of proliferation in stratified keratinocytes, we cultured HaCaT at the air–liquid interface for 12 days in the absence or presence of AVX001. We measured PGE2 levels and the thickness of the stratified epithelia. The proportions of proliferating and differentiating cells were determined based on Ki-67 and cytokeratin (CK) 10 positivity, respectively. AVX001-treated cultures had reduced levels of PGE2, indicating the compound maintains the ability to suppress PGE2 levels in stratified cultures (Figure 4A.) There was no significant difference in the thickness of the cultures (Figure 4B,E), however, immunohistochemical analysis of Ki67 showed strikingly fewer proliferating cells in the AVX001-treated cultures compared to the vehicle-treated controls (Figure 4C,E). This was not, however, accompanied by an increase in the proportion of CK10-positive cells, as might be expected, but rather by an accompanying reduction in the proportion of CK10-positive cells (Figure 4D,E). These findings give support for cPLA2α being primarily a regulator of keratinocyte proliferation. *Biomolecules* **2020**, *10*, x 10 of 18 this was inhibited by AVX001 (5 µM) (Figure 3D). These findings support cPLA2α as a target for inhibiting growth factor-dependent proliferation by halting cells in G1. AVX001 inhibited cell proliferation in HaCaT grown in monolayers; however, experiments performed in 3D culture systems are often considered to have more physiological relevance. HaCaT retain the ability to stratify and differentiate in culture [66,67], and this is dependent on the presence of exogenous growth factors [68]. To test whether AVX001 is an effective inhibitor of proliferation in stratified keratinocytes, we cultured HaCaT at the air–liquid interface for 12 days in the absence or presence of AVX001. We measured PGE2 levels and the thickness of the stratified epithelia. The proportions of proliferating and differentiating cells were determined based on Ki-67 and cytokeratin (CK) 10 positivity, respectively. AVX001-treated cultures had reduced levels of PGE2, indicating the compound maintains the ability to suppress PGE2 levels in stratified cultures (Figure 4A.) There was no significant difference in the thickness of the cultures (Figure 4B,E), however, immunohistochemical analysis of Ki67 showed strikingly fewer proliferating cells in the AVX001 treated cultures compared to the vehicle-treated controls (Figures 4C,E). This was not, however, accompanied by an increase in the proportion of CK10-positive cells, as might be expected, but rather by an accompanying reduction in the proportion of CK10-positive cells (Figure 4D,E). These findings give support for cPLA2α being primarily a regulator of keratinocyte proliferation.

**Figure 4.** AVX001 inhibited proliferation in a stratified epithelium. HaCaT grown on porous collagencoated membranes were maintained at the air–liquid interface for 12 days with exposure to exogenous EGF (1 ng/mL) in the absence or presence of AVX001 (5 µM). (**A**) PGE2 levels measured in the supernatants at day 12 by ELISA. (**B**–**D**) Immunohistochemistry using anti-Ki-67 MIB1 (DAB+) and anti-cytokeratin (CK)10 (Permanent Red) antibodies with hematoxylin counterstain. Brightfield images were taken at 40X magnification and used to quantify (**B**) the thickness (stratification) of the cultures, (**C**) the proportion of proliferating cells (proliferation index = Ki-67-positive cells/total cells), and (**D**) the proportion of CK10-positive cells (CK10 index = CK10 positive cells/total cells.) Typically, >20 images were collected at 40X magnification per replicate, with data being the mean ± SEM for three replicates. Statistical significance was calculated by one-way ANOVA with Dunnett's postanalysis; \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.005 versus vehicle-treated control. € Three representative images are shown for (i) vehicle and (ii) AVX001-treated cultures. **4. Discussion Figure 4.** AVX001 inhibited proliferation in a stratified epithelium. HaCaT grown on porous collagen-coated membranes were maintained at the air–liquid interface for 12 days with exposure to exogenous EGF (1 ng/mL) in the absence or presence of AVX001 (5 µM). (**A**) PGE2 levels measured in the supernatants at day 12 by ELISA. (**B**–**D**) Immunohistochemistry using anti-Ki-67 MIB1 (DAB+) and anti-cytokeratin (CK)10 (Permanent Red) antibodies with hematoxylin counterstain. Brightfield images were taken at 40X magnification and used to quantify (**B**) the thickness (stratification) of the cultures, (**C**) the proportion of proliferating cells (proliferation index = Ki-67-positive cells/total cells), and (**D**) the proportion of CK10-positive cells (CK10 index = CK10 positive cells/total cells.) Typically, >20 images were collected at 40X magnification per replicate, with data being the mean ± SEM for three replicates. Statistical significance was calculated by one-way ANOVA with Dunnett's post-analysis; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.005 versus vehicle-treated control. (**E**) Three representative images are shown for (i) vehicle and (ii) AVX001-treated cultures.

#### **4. Discussion**

In this study, we investigated the effects of the cPLA2α inhibitor AVX001 on inflammatory eicosanoid release and epidermal proliferation to understand its mode of action for treating psoriatic skin disease.

We demonstrate for the first time that AVX001 can significantly and dose-dependently suppress the production of both COX and LOX AA metabolites in stimulated human PBMC. The findings are consistent with the use of the cPLA2α inhibitors pyrrophenone and WAY-196025, which similarly inhibited both PGE2 and LTB4 release from PBMC stimulated with A23178 [68,69]. Our data thus support the fact that targeting the cPLA2α enzyme results in a balanced suppression of inflammatory eicosanoid release.

We further demonstrate the inhibitory effect of AVX001 on eicosanoid released in response to pro-inflammatory stimuli. The Toll-like receptor (TLR) 4 agonist LPS induced PGE2 release from PBMC, which was inhibited by AVX001. Our data using human PBMC supports the previously described involvement of cPLA2α in LPS-stimulated PGE2 production in THP-1 monocytes [35,70] reported to result from the induction of both the levels and activity of cPLA2α [5]. TNF-α is a pro-inflammatory cytokine and a key contributor to the pathogenesis of psoriasis [71]. TNF-α induced a robust production of PGE2 in HaCaT, which was inhibited by AVX001. These findings support cPLA2α as a mediator of the pro-inflammatory effects of TNF-α, as proposed by Sjursen et al. [36], however, while we demonstrated TNF-α-induced PGE2 production, Sjursen et al. reported that 6 h treatment with TNF-α preferentially induced HETE and not PGE2 production in HaCaT. It is therefore likely that stimulation of PGE2 release involves additional transcriptional upregulation of COX pathway enzymes in addition to cPLA2α activation in these cells, as demonstrated by Seo et al. [72].

Calcipotriol is a topical therapeutic for psoriasis and is known to cause skin irritation [54]. We demonstrate that calcipotriol stimulates the release of PGE2 both in PBMC and keratinocytes, which is in agreement with the following studies [55,56,73] and further supports the involvement of VDR/PGE2 signaling in drug-induced skin toxicity, as proposed by Shah et al. [56]. The mechanism by which calcipotriol stimulates PGE2 production is unclear. In AVX001-treated cells, calcipotriol was unable to stimulate PGE2 release, implicating that cPLA2α activation is required. This is in contrast to studies in keratinocytes by Ravid et al. [55], who suggest that the upregulation of COX-2 as opposed to increased AA production is responsible for the stimulation of PGE2 production. Doroudi et al. [74] present a VDR-independent mechanism for activation of cPLA2α by calcitriol via Ca2+/calmodulin-dependent protein kinase II (CAMKII)-dependent phosphorylation. It will be interesting to determine how PGE2 is regulated by calcipotriol in PBMC and keratinocytes and whether direct activation of cPLA2α by CAMKII is involved. For the treatment of psoriasis, calcipotriol is commonly combined with the potent corticosteroid betamethasone dipropionate (Daivobet), resulting in improved efficacy and tolerance [75]. This poses the possibility that the use of AVX001 could be an interesting non-steroidal alternative combination partner for reducing inflammation and improving tolerance to calcipotriol.

Our finding that EGF-stimulated PGE2 release in keratinocytes is reduced by inhibition of cPLA2α is in line with several reports linking EGF stimulation with AA release [76–79]. Furthermore, Naini et al. describe a requirement for intact cPLA2α/PGE2 signaling in growth factor-dependent cell cycle progression in both mouse embryonic fibroblasts (MEFs) and mesangial cells [38]. Collectively, this puts regulation of the cPLA2α enzyme, by means of its level and activity, in a central position to modulate growth factor-dependent responses. Thus, cPLA2α may control both inflammatory and mitogenic processes, which are hallmarks of the pathogenesis of psoriasis.

We further show that treatment with AVX001 inhibits EGF-stimulated S-phase entry and reduces the proliferation of HaCaT keratinocytes grown both in monolayers and stratified cultures. Our findings are in agreement with the established role of cPLA2α and eicosanoid signaling molecules as drivers of proliferation in several cancerous and non-cancerous cell types (reviewed in [80] and [81]). The described role of PGE2 as an autacoid growth factor [82,83] and effector of EGF responses in keratinocytes [84] make it a good candidate for mediating the effects of cPLA2α inhibition on keratinocyte proliferation. Knockout of the PGE2 receptor, EP2, also supports a role for PGE2 in regulating keratinocyte proliferation [85,86]. However, PGE2 is certainly not the only candidate, and a weakness of this study was our focus on the effects of AVX001 on AA metabolites. It is likely that cPLA2αinhibition with AVX001 would also suppress the production of LPC and its metabolites, e.g. platelet-activating factor (PAF). Like the eicosanoids, PAF has pro-inflammatory and proliferative effects in the epidermis [46,87,88], and PAF inhibition was found to suppress psoriasis-like skin disease progression in mice [89]. In future studies, it will therefore be important to determine whether AVX001 can also suppress the formation of LPC metabolites, as well as to determine which lipid mediators are the most critical effectors of keratinocyte proliferation under conditions of chronic inflammation.

#### **5. Conclusions**

In summary, we show that inhibition of cPLA2α with AVX001 inhibits eicosanoid release from primary human PBMC, limits the release of PGE2 in response to inflammatory mediators and EGF, and inhibits the proliferation of keratinocytes by preventing S-phase entry in response to growth factor stimulation. These findings suggest that the therapeutic mode of action of AVX001 in psoriasis could depend both on reducing inflammatory eicosanoid production and on inhibition of the hyperproliferative state of keratinocytes. We additionally propose that AVX001 could improve tolerance to calcipotriol, which could be relevant for developing a combination therapy to treat psoriasis.

**Author Contributions:** Conceptualization, B.J., A.J.F., and F.J.A.; methodology, A.J.M., F.J.A., N.M., and H.M.F.; software, F.J.A.; validation, F.J.A., N.M., and A.J.F.; formal analysis, F.J.A., and N.M.; investigation, F.J.A., N.M., and H.M.F.; resources, B.J. and A.J.F.; data curation, F.J.A.; writing—original draft preparation, F.J.A.; writing—review and editing, F.J.A., A.J.F., and B.J.; visualization, F.J.A. and A.J.F.; supervision, B.J., A.J.F., and F.J.A.; project administration, B.J. and A.J.F.; funding acquisition, B.J. and A.J.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Research Council of Norway grant no. 269792 and Coegin Pharma.

**Acknowledgments:** The immunohistochemistry was carried out at the Cellular and Molecular Imaging Core Facility (CMIC), Norwegian University of Science and Technology (NTNU), with the help of Ingunn Nervik. CMIC is funded by the Faculty of Medicine at NTNU and Central Norway Regional Health Authority.

**Conflicts of Interest:** B.J. is a shareholder of Coegin Pharma. A.J.F. and N.M. are employees of Coegin Pharma. The other authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

*Biomolecules* **2020**, *10*, 1402

#### **Appendix A**

*Biomolecules* **2020**, *10*, x 13 of 18

**Figure A1.** Inhibition of eicosanoid release in A23178-stimulated PBMC. Eicosanoid levels were measured in the supernatants of PBMC pre-incubated with vehicle, AVX001, naproxen, celecoxib, or NDGA in the range of 0.5–10 µM, and stimulated with A23178 (30 µM, 15 min). Data are the mean ± SEM for (**A**) PGE2, (**B**) LTB4, (**C**) TxB2, and (**D**) 12S-HETE from four individuals (PGE2, LTB4, TxB2) or three individuals (12S-HETE).  **Figure A1.** Inhibition of eicosanoid release in A23178-stimulated PBMC. Eicosanoid levels were measured in the supernatants of PBMC pre-incubated with vehicle, AVX001, naproxen, celecoxib, or NDGA in the range of 0.5–10 µM, and stimulated with A23178 (30 µM, 15 min). Data are the mean ± SEM for (**A**) PGE2 , (**B**) LTB4 , (**C**) TxB2, and (**D**) 12S-HETE from four individuals (PGE2 , LTB4 , TxB2) or three individuals (12S-HETE). Statistical significance was calculated using ANOVA with Dunnett's post-analysis, whereby % *p* < 0.1, # *p* < 0.05, ## *p* < 0.01 versus unstimulated control and ¤ *p* < 0.1, \* *p* < 0.05, \*\* *p* < 0.01 versus vehicle-treated control.

## **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Lipid Profile of Activated Macrophages and Contribution of Group V Phospholipase A2**

**Masaya Koganesawa, Munehiro Yamaguchi, Sachin K. Samuchiwal and Barbara Balestrieri \***

Department of Medicine, Division of Allergy and Clinical Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA<EMAIL_ADDRESS>(M.K<EMAIL_ADDRESS>(M.Y<EMAIL_ADDRESS>(S.K.S.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Macrophages activated by Interleukin (IL)-4 (M2) or LPS+ Interferon (IFN)γ (M1) perform specific functions respectively in type 2 inflammation and killing of pathogens. Group V phospholipase A2 (Pla2g5) is required for the development and functions of IL-4-activated macrophages and phagocytosis of pathogens. Pla2g5-generated bioactive lipids, including lysophospholipids (LysoPLs), fatty acids (FAs), and eicosanoids, have a role in many diseases. However, little is known about their production by differentially activated macrophages. We performed an unbiased mass-spectrometry analysis of phospholipids (PLs), LysoPLs, FAs, and eicosanoids produced by Wild Type (WT) and *Pla2g5*-null IL-4-activated bone marrow-derived macrophages (IL-4)BM-Macs (M2) and (LPS+IFNγ)BM-Macs (M1). Phosphatidylcholine (PC) was preferentially metabolized in (LPS+IFNγ)BM-Macs and Phosphatidylethanolamine (PE) in (IL-4)BM-Macs, with Pla2g5 contributing mostly to metabolization of selected PE molecules. While Pla2g5 produced palmitic acid (PA) in (LPS+IFNγ)BM-Macs, the absence of Pla2g5 increased myristic acid (MA) in (IL-4)BM-Macs. Among eicosanoids, Prostaglandin E2 (PGE2 ) and prostaglandin D2 (PGD2 ) were significantly reduced in (IL-4)BM-Macs and (LPS+IFNγ)BM-Macs lacking Pla2g5. Instead, the IL-4-induced increase in 20-carboxy arachidonic acid (20CooH AA) was dependent on Pla2g5, as was the production of 12-hydroxy-heptadecatrienoic acid (12-HHTrE) in (LPS+IFNγ)BM-Macs. Thus, Pla2g5 contributes to PE metabolization, PGE2 and PGD2 production independently of the type of activation, while in (IL-4)BM-Macs, Pla2g5 regulates selective lipid pathways and likely novel functions.

**Keywords:** Group V phospholipase A2 ; macrophages; lipids

### **1. Introduction**

Macrophages are heterogeneous cells that contribute to the pathogenesis of infectious disease, type 2 immune responses, and metabolic disorders [1–3]. Macrophages exposed to pathogen-associated molecular patterns (PAMPS), endogenously formed danger-associated molecular patterns (DAMPs), and to the microenvironmental milieu rearrange their repertoire of cytokines and chemokines and contribute to the immune responses [4,5]. In vitro, the immune responses of macrophages are simplified by LPS+Interferon (IFN)γ (M1) and Interleukin (IL)-4 (M2) polarization [6,7]. Although it is unlikely that macrophages in vivo exist in mutually exclusive phenotypes [2,4], the polarization paradigm has helped to dissect critical immune features of macrophages. M2 macrophages develop during type 2 inflammation, prompted by Type 2 cytokines including IL-4 and IL-13, and they are characterized by the production of CCL22, CCL17, Arginase-1, and Transglutaminase 2 (TGM2) [7–9]. M1 macrophages develop during infection and are equipped with an array of cytokines, including IL-12, TNFα, and reactive oxygen species (ROS), to fight pathogens. Metabolically, macrophage polarization is distinguished by activation of aerobic glycolysis in M1 macrophages, particularly when M1 activation is achieved by IFNγ, in addition to LPS [10] and fatty acid oxidation in M2 [11]. Fatty acids (FAs) are involved in the remodeling of membrane phospholipid (mPLs), macrophage development and polarization [12–14],

**Citation:** Koganesawa, M.; Yamaguchi, M.; Samuchiwal, S.K.; Balestrieri, B. Lipid Profile of Activated Macrophages and Contribution of Group V Phospholipase A2. *Biomolecules* **2021**, *11*, 25. https://doi.org/biom 11010025

Received: 30 November 2020 Accepted: 23 December 2020 Published: 29 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

and they are essential for phagocytosis by fully differentiated macrophages [14]. Furthermore, polyunsaturated fatty acids (PUFAs) are the precursors of eicosanoids, proor anti-inflammatory lipid mediators, which are generated through three main pathways, cyclooxygenase (COX), lipoxygenase (LOX), and monooxygenases like CYP450 (CYP). Even before the identification of macrophage polarization and their spectrum of phenotypes, stimuli like Granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 reportedly induced 5-lipoxygenase (5-LOX) activation in macrophages [15,16], the limiting step in the generation of Cysteinyl leukotrienes (CysLTs), major mediators of inflammation [17]. COX-derived Prostaglandin E2 (PGE2) and PGD2 may have proor anti-inflammatory functions associated with LPS or IL-4 immune responses [18–20]. CYP2J2-derived epoxyeicosatrienoic acids (EETs) have opposite roles in human monocytemacrophages depending on the activation state of the macrophages [21], while CYP omegaoxidase 20-hydroxy-eicosatetraenoic acid (20-HETE) and its metabolite 20-carboxy arachidonic acid (20CooH AA) may have anti-inflammatory properties due to the ability to activate Peroxisome Proliferator-Activated Receptor α (PPARα) and PPARγ [22] while also activating G Protein-Coupled Receptor 75 (GPR75) [23]. However, whether M1 or M2 activated macrophages differentially produce FAs and eicosanoids has not been fully elucidated, but is potentially critical to understand how lipids produced by macrophages may contribute alone or in combination to the many diseases involving activated macrophages.

The release of FAs and eicosanoids requires the activity of at least one of the phospholipases A2 (PLA2s) enzymes. PLA2s hydrolyze membrane phospholipids to generate FAs, including saturated fatty acids, monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) and their metabolites eicosanoids, and lysophospholipids (LysoPLs). As PLA2s have substrate specificities and cell-expression preferences, likely, activated macrophages would preferentially express one or more PLA2s and, thus, contribute to the generation of selective bioactive lipids. It is worth noting that the constitutive and ubiquitous group IVA cytosolic PLA2 (cPLA2α or Pla2g4a), which preferentially releases Arachidonic Acid (AA), is expressed more abundantly in M1 macrophages than in M2 macrophages [24]. Of the secretory PLA2 family members, group V PLA2 (Pla2g5) is expressed in macrophages [25] and other cells [26]. Several studies, including ours, have reported the presence of Pla2g5 mRNA and protein in bone marrow-derived macrophages (BM-Macs), peritoneal macrophages, human monocyte-derived macrophages, macrophagecell lines [27]. Pla2g5 can potentiate Pla2g4a activation and AA release in macrophages activated with Toll-Like Receptor (TLR) agonists [28] and is required for phagocytosis and killing of pathogens [27,29]. In vivo, its absence results in increased LPS inflammation and mortality following Candida infection [30,31]. On the other hand, Pla2g5 is induced by IL-4 in mouse and human macrophages [20,32–34] and is expressed together with TGM2 in macrophages present in human nasal polyps of subjects with chronic rhinosinusitis [20]. Furthermore, Pla2g5 contributes to M2 macrophage development and functions [20,33,34]. Indeed, in allergic type 2 inflammation, Pla2g5 potentiates inflammation and activation of target cells, including T-cells and innate lymphoid cells type 2 (ILC2s) through the generation of the linoleic acid (LA) and oleic acid (OA) [32,34]. However, the contribution of Pla2g5 to lipid generated during M1 and M2 polarization is still not known, but is potentially critical to account for the functions of M1 and M2 macrophages.

To investigate the lipid generated during macrophage polarization, we activated BM-Macs with IL-4 or LPS+IFNγ and analyzed FAs, PLs, LysoPLs, and eicosanoids by gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled to mass spectrometry (LC-MS). We found that macrophages activated by LPS+IFNγ or IL-4 rearrange their membrane phospholipid composition by decreasing phospholipid content, particularly Phosphatidylethanolamines (PE) during IL-4 activation and Phosphatidylcholine (PC) during LPS+IFNγ activation. Furthermore, in M2 macrophages, the absence of Pla2g5 increased PE 34:2, PE 36:2, PE 38:4, and PE 38:5, while in M1 macrophages, there was a significant increase in PC 34:1 and PC 36:2. LysoPLs were not significantly modified during macrophage activation in BM-Macs lacking Pla2g5. We

also found that Pla2g5 regulated the release of selective saturated fatty acids in activated BM-Macs. Furthermore, PGD2 and PGE2 were significantly induced by Pla2g5 in both M1 and M2 activation, while 20CooH AA production was dependent on Pla2g5 only in M2. Therefore, it is likely that the combined action of the lipids produced by M1 and M2 macrophages may contribute to the functions of macrophages in different settings. Identifying potential lipid signatures in M1 and M2 macrophages could uncover new pathways critical for the development, persistence, or reduction of inflammation. Some of these functions may rely on the presence or absence of Pla2g5 in macrophages.

#### **2. Materials and Methods**

#### *2.1. Macrophage Cultures*

BM-Macs were generated as previously described [34]. Briefly, BM cells were collected from femurs and tibiae of mice of Wild Type (WT) and *Pla2g5*-null mice [35]. The disaggregated cells were counted and suspended in complete medium (Dulbecco's Modified Eagle Medium (DMEM) F12, 5% Fetal Bovine Serum (FBS), 100 U mL−1 penicillin, 100 mg mL−1 streptomycin, 0.1 mM nonessential amino acids, 2 mM L-glutamine and 0.05 mM 2-mercaptoethanol (2-ME)) at a concentration of 4.0 × 106 cells per mL in 10 mL in 100 mm Petri dish. WT and *Pla2g5*-null BM-macrophages were cultured for 7 days in 50 ng mL−1 murine recombinant-Macrophage Stimulating Factor (rM-CSF) (PeproTech). Macrophages were activated with IL-4 or LPS+IFNγ (PeproTech) for 24 h, as previously described [32]. Supernatants and adherent cells were collected [20,34], frozen, and shipped for analysis by mass spectrometry.

Human monocyte-derived macrophages were generated as previously described [20]. Briefly, leukocyte-enriched buffy coat from healthy donors was overlaid on Ficoll-Paque Plus (GE Healthcare, Buckinghamshire, UK) and centrifuged at 600× *g* for 20 min. The mononuclear layer at the interface was collected, washed, and counted. Monocytes were isolated by negative selection (Miltenyi Biotec, Auburn, CA, USA) and plated at 2.2 × 105 cells/cm2 in 100 mm Petri dishes. Monocytes were cultured for 13 d in complete medium (RPMI 1640, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptavidin, 10% nonessential amino acids, 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1% sodium pyruvate, and 50 mM 2-ME supplemented with 50 ng mL−1 human rGM-CSF (R&D Systems, Minneapolis, MN, USA) [36]. To knock down PLA2G5, monocytes were cultured for 13 d in rGM-CSF, then they were transfected with human PLA2G5 ON-TARGET Plus SMART Pool siRNA or nontargeting vector ONTARGET Plus Control Pool (1000 nM; GE Dharmacon, Lafayette, CO, USA) as previously described [20]. To activate macrophages, cells were polarized for 24 h in complete medium, supplemented with 40 ng mL−1 human IL-4 (R&D Systems). Cell-free supernatants were collected, frozen and shipped for analysis by mass spectrometry.

#### *2.2. Mass Spectrometry of Lipids*

Lipid analysis was performed at the University of California San Diego Lipidomics Core [37]. For analysis of eicosanoids samples, the amount of sample used was 200 µL, and 100 µL internal standard was added. Lipids were extracted with Solid Phase Extraction (SPE): Strata-x polymeric reverse phase columns (8B-S100-UBJ; Phenomenex, Torrance, CA, USA). The following was added to each column: 100% MeOH, 100% H2O, sample, 10% MeOH, and 100% MeOH for elution. Samples were dried with a Speed-Vac (Thermo Scientific) and taken up in 100 mL buffer A (63% H2O, 37% acetonitrile, 0.02% acetic acid). Five microliters were injected into the ultra-high performance liquid chromatography system. The analysis was performed with a mass spectrometer (6500 Qtrap; Sciex, Framingham, MA, USA) [38]. For analysis of phospholipids, samples were extracted via Bleigh Dyer. Samples were dried down and taken up in 50 µL buffer A (59/40/1 isopropanol (IPA)/hexane (HEX)/H20 with 10 mM NH4OAC). Then, 10 µL were injected into UPLC-MS/MS. The first number of the phospholipids designates the total number of carbons and the second number after the colon indicates the total number of double bonds. For

example, PC 36:4 indicates a phospholipid molecule with a total of 36 carbons and 4 double bonds. Lysophosphatidylcholine (LysoPC)-O and PC-O indicate ether-linked fatty acids in the sn-1 position. We measure isobaric species. For example, PC 36:4 can consist of either PC (18:2; 18:2) or PC (16:0; 20:4). Similarly, we did not discriminate between 34:0 PC and 36:7 PC-O—both have identical molecular masses and are not separated by LC-MS.

FA analysis was performed according to a previously published method [39]. Briefly, the cell pellet was homogenized in 500 mL of PBS/10% methanol. An aliquot of 200 mL corresponding to about 0.5 × 106 cells was withdrawn and a cocktail of internal standards consisting of 15 deuterated fatty acids was added. The extraction was initiated with 500 mL of methanol and 25 mL of 1 N HCl and a bi-phasic solution is formed by addition of 1.5 mL of isooctane. The phases are separated by centrifugation and the isooctane phase containing the FFAs fraction was removed. The extraction is repeated once and the combined extracts were evaporated to dryness. The FFAs were derivatized with pentafluorobenzyl (PFB) bromide and the resulting fatty acid PFB esters were analyzed by gas chromatography/mass spectrometry using a negative chemical ionization mode (Agilent 6890N gas chromatograph equipped with an Agilent 5973 mass selective detector; Agilent, Santa Clara, CA, USA). Standard curves for each of the fatty acids were acquired in parallel using identical conditions. The quantitative assessment of fatty acids in a sample was achieved by comparison of the mass spectrometric ion signal of the target molecule normalized to the internal standard with the matching standard curve according to the isotope dilution method and by protein content.

#### *2.3. qPCR*

Total RNA was isolated from lysate with the RNeasy Micro Kit (Qiagen, Louisville, KY, USA), reverse transcribed into cDNA (High-Capacity cDNA Reverse Transcription Kit; Thermo ARTICLES science-Applied Biosystems, Foster City, CA, USA) and measured by real-time PCR with the use of SYBR® Green/ROX master mix (SABiosciences, Frederick, MD, USA) on an Mx3005P thermal cycler (Stratagene, Santa Clara, CA, USA). The ratio of each mRNA relative to the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mRNA was calculated with the ∆∆Ct threshold cycle method. The mouse primers used were GAPDH F: 50 -TCAACAGCAACTCCCACTCTTCCA-30 ; R: 50 -ACCCTGTTGCTGTAGCCGT ATTCA-300 Pla2g5 F: 50 -TGGTTCCTGGCTTGCAGTGTG-30 ; R: 50 -TTCGCAGATGACTAGG CCATT-30 [34]. Real-time PCR products were run on a 1.5% agarose gel and visualized using chemilmager 4400 fluorescence system (Alpha Innotech, Missouri, TX, USA).

#### *2.4. Statistical Analysis*

Comparisons between two groups were made by using an unpaired Student's *t* test, and other comparisons were made with two-way ANOVA with Tukey, Sidak, or Dunnett correction for multiple comparisons. Comparisons were performed with Prism software (GraphPad, San Diego, CA, USA). Data are expressed as mean ± standard error of the mean (SEM); significance was set at *p* < 0.05.

#### **3. Results**

#### *3.1. Phospholipid Metabolism in Bone Marrow-Derived Macrophages Activated by IL-4 or LPS+IFNγ*

To investigate the production of bioactive lipids in activated BM-Macs, we first asked whether, during macrophage activation, there were changes in the composition of membrane phospholipids (PLs), substrates of PLA2. We cultured mouse BM-Macs for 7 days in rM-CSF and 24 h with IL-4 or LPS+IFNγ, hereafter referred to as (IL-4)BM-Macs or (LPS+IFNγ)BM-Macs, respectively, for M2 or M1 (Figure 1a, inset), to distinguish them from other in vitro derived M2 or M1 macrophages [40]. The supernatants were collected and the phospholipid species analyzed by mass spectrometry. Five PL species were detected in resting WT BM-Macs. Phosphatidylcholine (PC) was the most abundant, followed by phosphatidylethanolamines (PE), while phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA) contributed minimally to membrane composition (Figure 1a, black columns). Compared to WT unstimulated (U)BM-Macs, PE was significantly decreased in WT (IL-4)BM-Macs, while PC was significantly reduced in (LPS+IFNγ)BM-Macs (Figure 1a). PC, PS, PI, and PA did not change following activation. PG species were undetectable. These data suggest that PE could be the prevalent substrate for a PLA2 in (IL-4)BM-Macs and PC in (LPS+IFNγ)BM-Macs, although other phospholipases could contribute to remodeling of membrane phospholipids. followed by phosphatidylethanolamines (PE), while phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA) contributed minimally to membrane composition (Figure 1a, black columns). Compared to WT unstimulated (U)BM-Macs, PE was significantly decreased in WT (IL-4)BM-Macs, while PC was significantly reduced in (LPS+IFN)BM-Macs (Figure 1a). PC, PS, PI, and PA did not change following activation. PG species were undetectable. These data suggest that PE could be the prevalent substrate for a PLA2 in (IL-4)BM-Macs and PC in (LPS+IFN)BM-Macs, although other phospholipases could contribute to remodeling of membrane phospholipids.

*3.1. Phospholipid Metabolism in Bone Marrow-Derived Macrophages Activated by IL-4*

To investigate the production of bioactive lipids in activated BM-Macs, we first asked whether, during macrophage activation, there were changes in the composition of membrane phospholipids (PLs), substrates of PLA2. We cultured mouse BM-Macs for 7 days in rM-CSF and 24 h with IL-4 or LPS+IFN, hereafter referred to as (IL-4)BM-Macs or (LPS+IFN)BM-Macs, respectively, for M2 or M1 (Figure 1a, inset), to distinguish them from other in vitro derived M2 or M1 macrophages [40]. The supernatants were collected and the phospholipid species analyzed by mass spectrometry. Five PL species were detected in resting WT BM-Macs. Phosphatidylcholine (PC) was the most abundant,

*Biomolecules* **2021**, *11*, x FOR PEER REVIEW 5 of 16

**3. Results**

*or LPS+IFN*

**Figure 1.** Phospholipid metabolism in bone marrow-derived macrophages (BM-Macs). Wild Type (WT) BM-Macs were Unstimulated (U) or stimulated with Interleukin (IL)-4 or LPS+ Interferon (IFN)γ for 24 h, supernatants were removed and collected for analysis by liquid chromatography coupled to mass spectrometry (LC-MS). (**a**) \* *p* < 0.05 of (U) vs. IL-4 or LPS+IFNγ BM-Macs. (**b**,**d**) Phosphatidylethanolamine (PE) molecules or (**c**,**d**) phosphatidylcholine (PC) molecules are identified by the binding of fatty acids which are described by the numbers of carbons and double bonds. PE-O and PC-O indicate ether linked fatty acids in the sn-1 position. The data are shown volcano plot of mean (**d**) or graph (**a**–**c**) of mean and standard error of three independent determinations. \*\*\* *p* < 0.0001, \*\* *p* < 0.005 (IL-4)BM-Macs reduction vs (U)BM-Macs; ### *p* < 0.0001, ## *p* < 0.005, # *p* < 0.05 (LPS+IFNγ)BM-Macs reduction vs. (U)BM-Macs; & *p* < 0.05 increase in (LPS+IFNγ)BM-Macs vs. (U)BM-Macs. *p*-values were obtained by two-way ANOVA with Dunnett's correction for multiple comparisons.

Next, we wanted to ascertain whether, among PE or PC species, there were preferred phospholipid molecules decreased during each of the macrophage activation models and, therefore, preferred bioactive lipid produced. We analyzed 69 PE molecules. In WT (U)BM-Macs, PE 36:2 was the most abundant. Compared to WT (U)BM-Macs, PE 34:1, PE 36:2, PE 38:4, and PE 38:5 were significantly decreased in both (IL-4) and (LPS+IFNγ)BM-Macs, and PE 34:2 was significantly reduced in (LPS+IFNγ)BM-Macs while PE 36:1 was significantly reduced in (IL-4)BM-Macs (Figure 1b). These data indicate that long-chain PUFAs, including AA, are liberated from PE in both IL-4 and LPS+IFNγ activation. Instead, PE-O 38:5 and PE 40:5 were significantly increased in WT (LPS+IFNγ)BM-Macs compared to WT (U)BM-Macs, likely indicating re-acylation. Of the 80 PC species analyzed (Figure 1c), PC 34:1 was the most abundant in WT (U)BM-Macs and was reduced in both (IL-4) and (LPS+IFNγ)BM-Macs as were PC 34:2, PC 36:1, PC 36:2 PC-O 34:0/PC-O 36:7. LPS+IFNγ activation resulted in the selective reduction of PC 30:0, PC 32:0, PC 32:1, and PC 38:2/PC-O 40:9 compared to (U)BM-Macs. A direct comparison of PE or PC molecules in (IL-4)BM-Macs versus (LPS+IFNγ)BM-Macs confirmed that PE species were preferentially reduced and likely metabolized in (IL-4)BM-Macs and PC species in (LPS+IFNγ)BM-Macs (Figure 1d). These results suggest that one or more PLA2 may hydrolyze preferred substrates in IL-4 or LPS+IFNγ activated macrophages.

Since Pla2g5 is induced by IL-4 [32,34] and prefers PE as a substrate at least in human monocyte-macrophages activated by IL-4 [33], we wanted to understand whether the changes in membrane phospholipid were due at least partially to Pla2g5, particularly in IL-4 BM-Macs. We confirmed that Pla2g5 is induced in mouse BM-Macs by IL-4, but not LPS+IFNγ (Figure 2a) as previously shown [32]. In *Pla2g5*-null BM-Macs, PE was significantly increased in unstimulated, IL-4 and LPS+IFNγ stimulated BM-Macs compared to equally treated WT BM-Macs (Figure 2b) while PC was similar in both WT and *Pla2g5* null BM-Macs independently of the activation state. Furthermore, compared to equally treated WT BM-Macs, the percentage increase of PE was 71.7 ± 21.1% in (IL-4)BM-Macs, 41.1 ± 2.4% in (U)BM-Macs and 59.7 ± 26.1% in (LPS+IFNγ)BM-Macs (data not shown). These data suggests that PE is the preferred substrate of Pla2g5 in (IL-4)BM-Macs and that the low expression level of Pla2g5 in (U)BM-Macs and (LPS+IFNγ)BM-Macs is sufficient to mobilize PE.

To understand whether Pla2g5 was active on selective PE or PC molecules, we analyzed PE and PC species in *Pla2g5*-null BM-Macs compared to WT BM-Macs. PE 36:1 (18:0, 18:1), PE 36:2 (18:0, 18:2), and PE 38:4 (18:0, 20:4) were increased in BM-Macs lacking Pla2g5 independently of the activation state. PE 34:1(16:0; 18:1), PE 34:2 (16:0,18:2), and PE 38:5 (16:0, 22:5; 18:1, and 20:4) were significantly increased in *Pla2g5*-null (IL-4)BM-Macs while PE 34:1 (16:0; 18:1) was increased in *Pla2g5*-null (LPS+IFNγ)BM-Macs, compared to their respective controls (Figure 2c). In the PC group, PC 30:2 and PC 36:2 were increased in *Pla2g5*-null (U)BM-Macs compared to WT (U)BM-Macs (Figure 2d), while PC 34:1 and PC 36:2 were increased in *Pla2g5*-null (LPS+IFNγ)BM-Macs compared to relative controls. These data suggest that Pla2g5 preferentially metabolized PE molecules in both (IL-4)BM-Macs and (LPS+IFNγ)BM-Macs and that its activity on PC is restricted to (LPS+IFNγ)BM-Macs.

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**Figure 2.** PE and PC molecules from WT and *Group V phospholipase A2 (Pla2g5)-*null BM-Macs determined by LC-MS. Expression of Pla2g5 mRNA relative to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) measured by qPCR in WT (black bars) or *Pla2g5*-null BM-Macs (grey bars), Unstimulated (U) or stimulated with IL-4 or LPS+IFN (**a**). BM-Macs isolated from WT (solid bars) or *Pla2g5-*null (dotted bars) mice were Unstimulated (U) or treated with IL-4 or LPS+IFN for 24 h and (**b**) total PC and PE, (**c**) PE, or (**d**) PC molecules are reported. The data are shown as a heatmap of mean or **Figure 2.** PE and PC molecules from WT and *Group V phospholipase A2 (Pla2g5)*-null BM-Macs determined by LC-MS. Expression of Pla2g5 mRNA relative to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) measured by qPCR in WT (black bars) or *Pla2g5*-null BM-Macs (grey bars), Unstimulated (U) or stimulated with IL-4 or LPS+IFNγ (**a**). BM-Macs isolated from WT (solid bars) or *Pla2g5*-null (dotted bars) mice were Unstimulated (U) or treated with IL-4 or LPS+IFNγ for 24 h and (**b**) total PC and PE, (**c**) PE, or (**d**) PC molecules are reported. The data are shown as a heatmap of mean or graph of mean and standard error of three independent determinations. # *p* < 0.0001, \*\* *p* < 0.005, \* *p* < 0.05 by two-way ANOVA with Sidak's or Tukey's correction for multiple comparisons.

#### *3.2. Lysophospholipid and Fatty Acid Generation in Activated BM-Macs 3.2. Lysophospholipid and Fatty Acid Generation in Activated BM-Macs*

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ANOVA with Sidak's or Tukey's correction for multiple comparisons.

To further investigate the changes in bioactive lipids during macrophage activation, we analyzed LysoPLs by LC-MS. A heat map of LysoPLs generated in BM-Macs showed that compared to unstimulated WT BM-Macs, LysoPC and Lysophosphatidylethanolamine (LysoPE) are substantially reduced following activation with either IL-4 or LPS+IFNγ (Supplemental Figure S1). This is likely due to LysoPL being rapidly metabolized or re-acylated into membrane phospholipids [41]. Furthermore, there was no significant difference between WT and *Pla2g5*-null BM-Macs in either LysoPE or LysoPC molecules in any stimulation (Supplemental Figure S1). To further investigate the changes in bioactive lipids during macrophage activation, we analyzed LysoPLs by LC-MS. A heat map of LysoPLs generated in BM-Macs showed that compared to unstimulated WT BM-Macs, LysoPC and Lysophosphatidylethanolamine (LysoPE) are substantially reduced following activation with either IL-4 or LPS+IFN (Supplemental Figure S1). This is likely due to LysoPL being rapidly metabolized or re-acylated into membrane phospholipids [41]. Furthermore, there was no significant difference between WT and *Pla2g5-*null BM-Macs in either LysoPE or LysoPC molecules in any stimulation (Supplemental Figure S1).