Supplemental Material

Maresin 1 Activates LGR6 Promoting Phagocyte Immunoresolvent Functions

Nan Chiang, Stephania Libreros, Paul C. Norris, Xavier de la Rosa, Charles N. Serhan

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

1 Supplemental Table 1. Screening of orphan GPCRs with MaR1

Vehicle Vehicle MaR1 MaR1 mean RLU > GPCR ID SD % Activity Mean RLU Mean RLU + 2 SD Mean RLU Vehicle mean RLU+2 SD? ADMR 930920 33283 997486.5381 863760 -7% BAI1 172580 18362 209304.1828 176160 2% BAI2 26390 1354 29097.71737 26240 -1% BAI3 18040 758 19555.07976 18460 2% CCRL2 15090 402 15893.6583 13840 -8% CMKLR2 30080 1744 33568.954 28240 -6% DARC 119110 4817 128743.8016 126260 6% EBI2 101200 6004 113207.8197 105640 4% GHSR1B 3940 203 4345.298244 3700 -6% GPR101 41740 1593 44926.97349 41580 0% GPR103 21413 1484 24381.25067 23920 12% NO GPR107 366800 11007 388814.4922 360020 -2% GPR12 77980 1563 81105.4653 76260 -2% GPR123 1485190 46446 1578081.986 1342640 -10% GPR132 860940 17473 895885.901 826560 -4% GPR135 18720 1656 22032.6827 17540 -6% GPR137 40973 2285 45544.0809 39140 -4% GPR139 438280 16736 471751.0542 413120 -6% GPR141 30180 2080 34339.2307 29020 -4% GPR142 105250 12089 129427.069 101020 -4% GPR143 89390 5260 99910.40557 89380 0% GPR146 16860 551 17961.75617 16240 -4% GPR148 6160 484 7128.848113 7520 22% YES GPR149 50140 934 52008.76073 49720 -1% GPR15 10110 1086 12282.67884 10300 2% GPR150 36470 2724 41918.3759 36340 0% GPR151 52730 3187 59104.07248 47340 -10% GPR152 141950 9317 160584.4913 138140 -3% GPR157 358200 14714 387627.8236 343680 -4% GPR161 8050 1129 10308.377 7220 -10% GPR162 14750 1297 17344.25005 15120 3% GPR17 1085570 20872 1127313.158 1045800 -4% GPR171 8100 601 9301.776463 9000 11% NO GPR173 87030 3746 94522.25378 81220 -7% GPR176 431010 17476 465962.1196 413620 -4% GPR18 3320 393 4106.553664 3220 -3% GPR182 749570 28457 806483.1034 714820 -5% GPR20 135610 4104 143817.2813 127200 -6% GPR23 1203410 43969 1291348.072 1204500 0% GPR25 4290 826 5942.956946 4500 5% GPR26 2160 287 2733.178274 2180 1% GPR27 163370 6480 176330.0617 159940 -2% GPR3 1808630 44214 1897058.155 1811960 0% GPR30 7130 688 8506.953158 6640 -7% GPR31 6130 388 6906.659514 5960 -3% GPR32 139240 5586 150411.392 141060 1% GPR37 993010 16495 1025999.138 973600 -2% GPR37L1 56170 2469 61107.74577 55060 -2% GPR39 469030 31072 531174.4125 456860 -3% GPR4 8760 1326 11411.69128 8200 -6% GPR45 925030 20094 965217.3147 926960 0% GPR50 792240 26238 844715.8941 829460 5% GPR52 8960 571 10101.46105 10060 12% NO GPR6 57570 1683 60936.58086 61040 6% GPR61 240140 15202 270544.8768 225800 -6% GPR65 73000 2702 78403.80113 68380 -6% GPR75 95540 6715 108969.4403 95760 0% GPR78 59770 2998 65765.33152 62160 4% GPR79 65520 1042 67603.07465 64880 -1% GPR81 485040 11086 507212.6558 486100 0% GPR83 91630 4012 99654.86137 89920 -2% GPR84 50450 1619 53688.68286 47200 -6% GPR85 10650 571 11791.46105 9740 -9% GPR88 27850 4137 36123.55627 25580 -8% GPR91 15520 1710 18940.40933 17840 15% NO GPR97 1006310 27047 1060403.977 989660 -2% LGR4 13700 2273 18246.41251 13560 -1% LGR5 5720 510 6740.326745 5040 -12% LGR6 16600 1130 18859.9115 19520 18% YES MRGPRD 11810 1431 14672.44651 11220 -5% MRGPRE 399420 13490 426400.4868 399320 0% MRGPRF 645150 22893 690936.7696 616940 -4% MRGPRX1 2179150 41244 2261637.058 2162000 -1% MRGPRX4 140390 7526 155442.2468 135940 -3% OPN5 13950 1064 16078.03509 13300 -5% OXGR1 187130 5028 197185.075 185060 -1% P2RY8 875740 12164 900068.1127 844400 -4% TAAR5 2430 229 2888.984386 2030 -16% Using b-arrestin PathHunter GPCR system, a panel of orphan GPCRs (77 GPCRs) was screened in the presence of 10 nM of MaR1 or vehicle control (0.1 % ethanol). The % activity = 100% x (mean RLU of test sample – mean RLU of vehicle control) / (mean RLU of vehicle control). RLU, relative luminescence units. Candidates were selected based on the criteria that the mean RLU in the presence of MaR1 is larger than the mean RLU +2 SD with vehicle alone

2 Supplemental Table 2. Screening of known GPCRs with MaR1

GPCR ID % Activity GPCR ID % Activity GPCR ID % Activity GPCR ID % Activity

ADCYAP1R1 0% CMKLR1 0% GIPR -7% NPY1R -1%

ADORA3 -1% CMKOR1 -3% GLP1R 0% NPY2R 0%

ADRA1B -3% CNR1 -6% GLP2R -2% NTSR1 0%

ADRA2A 0% CNR2 0% GPR1 0% OPRD1 -1%

ADRA2B 0% CRHR1 -1% GPR109A -2% OPRK1 2%

ADRA2C 0% CRHR2 0% GPR119 -1% OPRL1 -1%

ADRB1 0% CRTH2 0% GPR120 11% OPRM1 0% ADRB2 0% CX3CR1 -10% GPR35 0% OXTR 0%

AGTR1 0% CXCR1 -1% GPR55 2% P2RY11 -1%

AGTRL1 0% CXCR2 0% GPR92 -3% P2RY12 -3%

AVPR1A -1% CXCR3 -2% GRPR 1% P2RY2 -2%

AVPR1B 1% CXCR4 11% HCRTR1 0% P2RY4 -10%

AVPR2 -1% CXCR5 -3% HCRTR2 0% P2RY6 -1%

BDKRB1 -1% CXCR6 0% HRH1 0% PPYR1 (NPY4) -2%

BDKRB2 -1% DRD1 0% HRH2 -2% PRLHR -4%

BRS3 5% DRD2L 0% HRH3 -1% PROKR1 -1% C5AR1 0% DRD2S -2% HTR1A -2% PROKR2 -1%

C5LR2 -6% DRD3 0% HTR1B -4% PTAFR 1% CALCR 1% DRD4 -10% HTR1E 3% PTGER2 1% CALCR + RAMP2 0% DRD5 0% HTR1F 2% PTGER3 0% CALCR + RAMP3 -7% EDG1 0% HTR2A 4% PTGER4 0%

CALCRL + RAMP1 0% EDG2 -6% HTR2C -3% PTGIR -1% CALCRL + RAMP2 -1% EDG3 -2% HTR5A -4% PTHR1 0% CALCRL + RAMP3 0% EDG4 0% KISS1R 3% PTHR2 0% CCKAR 0% EDG5 -2% LHCGR 8% RXFP3 1% CCKBR 2% EDG6 -1% LTB4R -1% SCTR -1% CCR10 -1% EDG7 -1% MC1R -14% SSTR2 0% CCR2 -1% EDG8 -8% MC3R -1% SSTR3 2% CCR3 4% EDNRA 1% MC4R -3% SSTR5 -1% CCR4 -1% EDNRB 0% MC5R -3% TACR1 -4% CCR5 -1% F2R -2% MCHR1 -2% TACR2 -2%

CCR6 0% F2RL1 0% MCHR2 -1% TACR3 0% CCR7 0% F2RL3 0% MLNR 0% TBXA2R -1% CCR8 -1% FPR1 0% MRGPRX2 -3% TRHR -1% CCR9 -1% FPRL1 0% MTNR1A 0% TSHR -2% CHRM1 -3% FSHR 2% MTNR1B 0% UTR2 -1% CHRM2 1% GALR1 0% NMU1R -2% VIPR1 0% CHRM3 -1% GALR2 -3% NPBWR1 -1% VIPR2 0% CHRM4 -3% GCGR -1% NPBWR2 -1% CHRM5 6% GHSR1A -2% NPFFR1 3%

Using b-arrestin PathHunter GPCR system, a panel of known GPCRs (158 GPCRs) was screened in the presence of 10 nM of MaR1 or vehicle control (0.1 % ethanol). The % activity = 100% x (mean RLU of test sample – mean RLU of vehicle control) / (mean RLU of vehicle control). RLU, relative luminescence units.

3 Supplemental Table 3. AA, EPA and DHA bioactive metabolome

LM levels (pg/ml)

AA bioactive Q1 Q3 Mock transfection LGR6 transfection Mock transfection + E. coli LGR6 transfection + E. coli metabolome LXA 4 351 115 18.5 ± 12.6 15.6 ± 10.7 24.9 ± 17.3 19.0 ± 13.3 LXB 4 351 221 1.6 ± 1.1 1.3 ± 0.9 2.3 ± 1.9 0.9 ± 0.6 AT-LXA 4 351 115 16.1 ± 10.2 26.8 ± 17.8 28.1 ± 15.0 29.0 ± 14.5 AT-LXB 4 351 221 102.4 ± 46.7 115.4 ± 53.8 85.1 ± 41.4 101.5 ± 46.7 LTB 4 335 195 11.8 ± 6.8 9.9 ± 5.6 56.4 ± 47.4 60.5 ± 55.9 PGD 2 351 233 54.8 ± 22.0 55.7 ± 19.6 70.7 ± 27.9 73.7 ± 27.5 PGE 2 351 189 51.3 ± 16.3 51.2 ± 14.4 58.4 ± 22.2 59.2 ± 16.7 PGF 2α 353 193 129.6 ± 43.0 104.8 ± 29.9 155.9 ± 48.0 111.7 ± 35.7 TXB 2 369 169 2757.6 ± 735.8 2935.5 ± 805.6 3894.9 ± 1151.8 3048.0 ± 924.5

DHA bioactive metabolome

RvD1 375 121 - -

RvD2 375 175 - -

RvD3 375 147 9.9 ± 2.3 9.0 ± 2.0 9.9 ± 1.5 10.7 ± 2.1 RvD4 359 255 - -

RvD5 359 199 2.7 ± 0.7 2.3 ± 0. 4 3.9 ± 0. 8 2.7 ± 0.6 RvD6 - - AT-RvD1 375 121 7.0 ± 5.6 6.9 ± 4.7 6.2 ± 5.8 5.8 ± 4.2 AT-RVD3 375 147 0.6 ± 0.4 0.4 ± 0.2 0.7 ± 0.4 0.9 ± 0.6

PD1 359 153 0.4 ± 0.1 0.2 ± 0.1 0.6 ± 0.3 0.3 ± 0.1 AT-PD1 359 153

MaR1 359 221 4.2 ± 2.2 5. 2 ± 2.5 5. 4 ± 2.7 3. 5 ± 1.2

EPA bioactive metabolome

RvE1 349 195 - - - - RvE2 333 253 3.3 ± 1. 5 3. 8 ± 1. 1 1.0 ± 0. 8 3.4 ± 1. 8 RvE3 333 201 19.3 ± 4.0 21.1 ± 4.7 23.4 ± 4.5 24.3 ± 5.6

Human (1x106 cells) were transfected with mock or LGR6 (see Methods) for 3 days, followed by addition of E. coli (50x106 CFU) for 1h. Incubations were terminated by addition of 1V methanol with internal standards and extracted. LM levels were then determined using LM metabolopidomics (see Methods for details). Values are express as pg/ml (0.5x106 cells); mean ± SEM.

4 Supplemental Table 4. Sequences for shRNA knockdown*

Gene shRNA.name SplashRNA LGR6 97mer.construct ID Human 59352 LGR6_59352_1160 1.503 TGCTGTTGACAGTGAGCGACCTGTGAGTACCTCTTT GAAATAGTGAAGCCACAGATGTATTTCAAAGAGGTA CTCACAGGGTGCCTACTGCCTCGGA 59352 LGR6_59352_454 1.495 TGCTGTTGACAGTGAGCGACAGGAGTTTCCAGATCT CAAATAGTGAAGCCACAGATGTATTTGAGATCTGGA AACTCCTGGTGCCTACTGCCTCGGA 59352 LGR6_59352_1743 1.357 TGCTGTTGACAGTGAGCGACGGTGCCTACATCAAAC TGTATAGTGAAGCCACAGATGTATACAGTTTGATGTA GGCACCGGTGCCTACTGCCTCGGA 59352 LGR6_59352_453 1.204 TGCTGTTGACAGTGAGCGCCCAGGAGTTTCCAGATC TCAATAGTGAAGCCACAGATGTATTGAGATCTGGAA ACTCCTGGATGCCTACTGCCTCGGA 59352 LGR6_59352_1159 1.093 TGCTGTTGACAGTGAGCGACCCTGTGAGTACCTCTT TGAATAGTGAAGCCACAGATGTATTCAAAGAGGTAC TCACAGGGCTGCCTACTGCCTCGGA 59352 LGR6_59352_825 0.933 TGCTGTTGACAGTGAGCGCTGGGGGCTTGATGCATC TGAATAGTGAAGCCACAGATGTATTCAGATGCATCAA GCCCCCAATGCCTACTGCCTCGGA 59352 LGR6_59352_1698 0.829 TGCTGTTGACAGTGAGCGACGTGGCCCTGGTGATG ATGAATAGTGAAGCCACAGATGTATTCATCATCACCA GGGCCACGGTGCCTACTGCCTCGGA 59352 LGR6_59352_6 0.793 TGCTGTTGACAGTGAGCGCCAGCATGAACAACCTCA CAGATAGTGAAGCCACAGATGTATCTGTGAGGTTGT TCATGCTGATGCCTACTGCCTCGGA 59352 LGR6_59352_1749 0.744 TGCTGTTGACAGTGAGCGACTACATCAAACTGTACT GTGATAGTGAAGCCACAGATGTATCACAGTACAGTT TGATGTAGGTGCCTACTGCCTCGGA 59352 LGR6_59352_1704 0.700 TGCTGTTGACAGTGAGCGACCTGGTGATGATGAACT CCTTTAGTGAAGCCACAGATGTAAAGGAGTTCATCAT CACCAGGGTGCCTACTGCCTCGGA Control Ren.713 Negative Control TGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTAT CTATAGTGAAGCCACAGATGTATAGATAAGCATTATA ATTCCTATGCCTACTGCCTCGGA

* obtained using software in ref 50.

5

Supplemental Table 5. Antibodies used in cyTOF

Target epitope Clone Isotopic label Supplier pAKT (S473) D9E Sm 152 Fluidigm p-p38 (T180/Y182) D3F9 Gd 156 Fluidigm pStat3 (Y705) 4 Gd 158 Fluidigm pStat1 (Y701) 58D6 Gd 160 BWH pNF-kB p65 (S529) K10-895.12.50 Er 166 Fluidigm pERK (T202/Y204) D13.14.4E Er 167 Fluidigm pS6 (S235/S236) N7-548 Yb 172 Fluidigm HLA-DR L243 Yb 173 Fluidigm pStat5 (Y694) D47/E7 XP Yb 174 BWH pCREB (S133) 8763 Yb 176 Fluidigm

6 Supplemental Figure 1 A Vehicle curve

80 ### 60 ## 50 40 # RLU 20 ** 40 ** 0 4°C 25°C 37°C 40°C ** 30 Temperatures # 20 40°C 10 37°C

% increase (RLU) 0 25°C 4°C -10

-20 -10 -9 -8 MaR1, Log [M] B Vehicle curve 120 100 80 60 RLU 40 60 20 0 6.5 7.5 8.5 40 ** pH values pH 6.5 pH 7.5 20 pH 8.5

% increase (RLU) 0

-20 -10 -9 -8 MaR1, Log [M] C GPR32 ALX ALX GPR32 ## 5050 50 ## *** # ** 4040 40 ** *** *** * *** 3030 30 *

2020 % increase 20 % increase % % increase % increase % RvD1 RvD1RvD1 1010 10

MaR1 MaR1MaR1 00 00 -140 10-13-13 -1210-12 -1110-11 -1010-10 10-9-9 10-8-8 10-7-7 -140 10-13-13 -1210-12 -1110-11-1010-10 10-9-9 10-8 -8 10-7 -7 CompoundLog [M] [M] CompoundLog [M] [M] Supplemental Figure 1. MaR1 interactions with LGR6, ALX and GPR32 (A, B) MaR1-LGR6 interactions were monitored using CHO-b-arrestin-human LGR6 reporter cells under different temperatures (A, replotted from Figure 1F for direct comparison) and pH (B). Results are mean±SEM from 3 independent experiments and 3 replicates each experiment. (insets) vehicle curves under different temperatures and pH. (A) #P<0.05, ##P<0.01, ###P<0.001 vs 4°C, **P<0.01, vs 4°C and 25°C. (B) **P<0.01, pH 6.5 vs pH 8.5. (C) MaR1 interactions with HEK-b-arrestin-ALX and CHO-b-arrestin-human GPR32 system, compared to RvD1. Results are mean±SEM from 3 independent experiments and 3-4 replicates for each experiment. *P<0.05, **P<0.01, ***P<0.001, vs vehicle control; #P<0.05, ##P<0.01, vs MaR1. All panels were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Supplemental Figure 2 A PMN (13.2%) Monocytes LymphocytesLymphocytes

Isotype

LGR6 SSC-A Lymphocytes Monocytes LGR6 LGR6 SSC-A LGR6 SSC-A (21.5%) (1.95%) SSC-A

B M1 M2

FSC-A FSC-A

FSC-A FSC-A Isotype M1 A A

- M2 - M2 SSC-A SSC-A SSC-A SSC-A SC SC SSC-A SSC-A S SSC-A SSC-A S

FSC-A FSC-A FSC-A FSC-A FSC-A FSC-A FSC-A LGR6 FSCFSC-A-A FSC-A

CD80 CD54 CD206 CD163 M1 M2

C M1 M2

Ionophore Flux

(1µM) 2+ Ca

LTD4 (10nM) Flux 2+ 2+ Ca

MaR1 (10nM) Flux 2+ 2+ Ca

D Neutrophils Monocytes Lymphocytes

LTB4

MaR1 Flux 2+ 2+ Ca

Time

Supplemental Figure 2. Human leukocytes: LGR6 expression and intracellular calcium release (A) LGR6 expression in human peripheral blood leukocytes (PMN, monocytes and lymphocytes) was demonstrated using a rabbit anti-human LGR6 Ab by flow cytometry. (B) LGR6 expression in M1 (CD80, CD54) and M2 (CD206, CD163) macrophages (C, D) Calcium influx in (C) M1, M2 macrophages, and (D) peripheral blood leukocytes (PMN, monocytes and lymphocytes) was monitored using flow cytometry with a Indo-1 fluorescent indicator. Supplemental Figure 3 A B

) 40

) 0 MaR1-ME Ω Ω

-5 MaR1 30

-10 20

-15 10 Impedance change ( Impedance change ( -20 0 0 30 2 604 90 6 120 8 MaR1-ME MaR1 TimeTime (min)

C D

80 LTB4 ) 80

Ω EC50 ~ 0.12 nM 100 nM 60 60 10 nM 40 1 nM 40 100 pM 20 10 pM 20

Impedance Chamge 1 pM Impedance change ( 0 0 0 00 30 2 60 4 90 6 120 8 15010 18012 -12 -11 -10 -9 -8 -7 Time (min) Log [M]

E F 80

) 80

Ω *** 60 60 LTB 40 4 40 ### 20 20 MaR1 ###

Impedance Chamge PD1 0 0 vehicle 0 30 60 90 120 150 180 Impedance change ( 0 2 4 6 8 10 12 -20 Vehicle MaR1 PD1 LTB4 Time (min) 100 nM

Supplemental Figure 3. Human recombinant LGR6 and BLT1 receptor specificity (A, B) CHO-LGR6 cells were plated onto 8-well ECIS arrays (8W10E+), incubated with MaR1 (100 nM), MaR1-ME (100 nM) or vehicle alone (control), and impedance changes across CHO cell monolayers were continuously recorded every 4 seconds for 8 min using ECIS. Results are mean (A) or mean±SEM (B) of impedance changes; n=5. No statistical significance between MaR1 and MaR1-ME; two-tailed paired Student’s t-test.

(C-F) CHO-BLT1 cells were plated onto 8-well ECIS arrays (8W10E+), incubated with (C,D) LTB4

(0.001-100 nM), (E,F) MaR1, PD1, LTB4 (100 nM) or vehicle alone (control), and impedance changes across CHO cell monolayers were continuously recorded every 4 seconds using ECIS. Results are ### mean (C,E) or mean±SEM (D,F) from n=3, ***P<0.001, vs vehicle; P<0.001, vs. LTB4; one-way ANOVA with Tukey’s multiple comparisons test. 3500 Supplemental Figure 4 A Human dermal fibroblast 3000 MaR1 1200 (Ω)

2500 800 MaR1 12E-MaR1 400

2000 Impedance 0 MaR1 Maximal changes in 12E-MaR1 MaR1 2500 CHO-LGR6Vehicle

1500 12E-MaR1 100 nM 2000 Vehicle 10 nM 1000 1 nM 1500 vehicle

Changes in Impedance (Ω) 500 12E-MaR1 1000

0 0 2 4 6 8 Increase of impedance 10500 12 14 16 Time (h) 0 0 6 12 18 24 30 36 B D CHO-GFP-LGR6 CHO-GFP-mock CHO-LGR6 25002500 25002500 CHO-GFP 100 nM 20002000 10 nM 20002000 1 nM 100 nM 15001500 vehicle 15001500 10 nM

of impedance of 1 nM 10001000 10001000 vehicle Increase Increase of impedance Increase of impedance Increase of impedance 500500 500500

00 00 0 6 12 18 24 30 36 00 16 122 18 3 244 305 36 6 0 1 2 3 4 5 6 Time (h) Time (h)

2500 CHO-GFP

C 2000 150150 * * CHO-GFP-LGR6 1500 100 nM 10 nM CHO-GFP-mock 100100 1 nM 1000 vehicle

Increase of impedance 500 5050

0 % increase above vehicle above increase % 0 6 12 18 24 30 36 % increase above vehicle 0 0

0 1 1010 100 nM

Supplemental Figure 4. MaR1 stimulates fibroblasts wound repair stereoselectively and epithelial cells in a LGR6-dependent manner (A) Human dermal fibroblasts, (B-D) GFP-LGR6 or GFP-mock vector transfected CHO cells were wounded (1,250 mA, 64KHz, 30 seconds). Changes of impedance (Ω) was recorded every 10 min using ECIS (6h at 37 ºC) on 8W1E electrode arrays following addition of (A) 10 nM MaR1, 12E-MaR1 or vehicle control; (B-D) 1-100 nM MaR1 or vehicle control. Results are (A, B, D) representative real-time tracings from n=3-4; (A, inset) mean of n=3; (C) mean±SEM; n=3-4. *P<0.05, CHO-GFP-LGR6 vs CHO-GFP-mock; two-way ANOVA with Tukey’s multiple comparisons test. Supplemental Figure 5 A

IL-8 + vehicle + MaR1 1 nM

B C 120 100 * 50 * 100 40 * 75 80 30 * 60 50 * 20 40 * relative to IL-8

% of 25

10 Accumulated distance 20 % reduction of chemotaxis 0 0 0

IL-8 +Veh. +Veh. Vehicle

MaR1 10nM +MaR1 1nM +MaR1 1nM +MaR1 10nM +MaR1 10nM + IL-8 + IL-8

Mouse IgG D Rabbit IgG

IgG1 isotype control Nonnon-immune-immune IgG IgG LGR6 IgG1 LGR6LGR6 IgG IgG 50 50

### ### 40 *** *** 40 # *** ### *** *** *** 30 ** 30 * * ** ** % reduction % reduction 20 20 *

10 10

0 0 0 0.1 nM 1 nM 10 nM 100 nM 0 0.1 nM 1 nM 10 nM 100 nM MaR1 MaR1

Supplemental Figure 5. LGR6-mediated MaR1 actions on human PMN (A-C) PMN chemotaxis. Freshly isolated human PMN were incubated with 1 or 10 nM MaR1 or vehicles (PBS + 0.1% ethanol) for 10 minutes. Chemotaxis toward a IL-8 (10nM) gradient (10nM) in Ibidi Chemotaxis μ-Slide was monitored in real time for 2h. (A) Representative rose plots from 2 representative donors. (B) % chemotaxis and % reduction of IL-8-initiated chemotaxis; mean±SEM, n=4. *p<0.05, vs. IL-8 + vehicle. (C) Accumulated distance relative to IL-8 (100%); mean±SEM, n=3. *p<0.05, vs. IL-8. One-way ANOVA. (D) PMN transmigration. IL-8 (10 nM, 30 µl) were placed in the bottom chamber of the Neuro Probe chemotaxis chambers. CFSE-lableled PMN (5x106/ml) was incubated with mouse anti- human LGR IgG1, rabbit anti-human LGR6 IgG or isotype controls (1:100 dilutions) for 30 min at RT, followed by addition of MaR1 (0.1-100 nM) or vehicle for 15 min. PMN (25 µl) were then

added to the top the filters to initiate chemotaxis toward IL-8 for 2h (37°C in 5% CO2). Migrated cells in the bottom chambers were quantified using fluorescence. Results are mean±SEM, n=3. *p<0.05, **p<0.01, ***p<0.001, vs vehicle controls; #p<0.05, ###p<0.001, vs. isotype controls (mouse) or non-immune IgG (rabbit); two-way ANOVA with Tukey’s multiple comparisons test. Supplementary Figure 6

Donor 2 Donor 3 Donor 4

200000 200000 25000 LGR6, MaR1 LGR6, MaR1 LGR6, MaR1 LGR6, veh LGR6, veh LGR6, veh 20000 150000 150000

15000

100000 MFI MFI 100000 MFI 10000 Mock, MaR1 Mock, veh 50000 50000 Mock, MaR1 5000 Mock, MaR1 Mock, veh Mock, veh 0 0 0 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min) Time (min)

Rate (MFI/min) Rate (MFI/min) Rate (MFI/min) LGR6, MaR1 295 LGR6, MaR1 3013 LGR6, MaR1 2442 1364 LGR6, veh 221 200000 LGR6, veh 2206 150000 LGR6, veh 20000 Mock, MaR1 216 Mock, MaR1 1593 Mock, MaR1 2703 Mock, veh 140 Mock, veh 1219 15000 150000 Mock, veh 1956 100000

MFI 10000 100000 MFI MFI

50000 50000 5000

0 0 0 20 30 40 50 60 20 30 40 50 60 20 30 40 50 60 Time (min) Time (min) Time (min)

Supplemental Figure 6. Overexpression of human LGR6 enhances MaR1 actions in stimulating phagocytosis Human MF were transfected with human LGR6 or mock plasmid. Phagocytosis was carried out as in Figure 4A. Results are mean fluorescence intensity (MFI) (top panels) and kinetics of phagocytosis (bottom panels) from 3 separate donors. Supplemental Figure 7 A B

Docosahexaenoic acid derived MaR1 +H RvD2 RvD5 100 221 141 359 -H OH 246 OH -H +H 359=M-H 250 113 341=M-H-H2O 315=M-H-CO2 297=M-H-H2O-CO2 279=M-H-2H O-CO derived Protectins and 2 2 COO- 232=250-CO Maresins 2 MaR1 228=246-H2O 203=221-H2O 159=221-H O-CO 297 2 2 123=141-H2O Relative Intensity (%)

279 341 113 141 246 159 221 Eicosapentaenoic acid derived Resolvins 123 250 315 RvE3 0 RvE1 RvE2 100 140 180 220 260 300 340 380 m/z, Da

RvD5

100 359 derived 113 COO- 246 +H 261 5S,15S-diHETE HO +H -H 141 359=M-H AT-LXA4 341=M-H-H2O LXA4 HO 214 323=M-H-2H O AT-LXB4 2 297=M-H-H O-CO LXB4 2 2 279=M-H-2H2O-CO2 243=261-H2O 228=246-H2O 199=217-H2O Arachidonic acid derived Prostaglandins and Leukotrienes PGF 141 PGD2 2a PGE2 LTB4 TxB Relative Intensity (%) 199 341 2 297 261 279 113 323 100 140 180 220 260 300 340 380 0

Relative Abundance m/z, Da

0 2 4 6 8 10 12 14 16 18 20 22 Time, min

C MaR1 Total SPM

15 400

10 300

5 200 pg/ml pg/ml

0 100

-5 0 Mock LGR6 OE Mock LGR6 OE Mock LGR6 OE Mock LGR6 OE

Media + E. coli Media + E. coli

Supplemental Figure 7. LM/SPM metabololipidomics of human macrophages: LGR6 overexpression with E. coli incubation. Human MF were transfected with human LGR6 plasmid or a mock plasmid. MF (1x106 cells) were then incubated with E. coli (5x107 CFU) for 1h. Incubations were terminated using ice-cold methanol, extracted and LM/SPM levels determined using mass-spectrometry-based metabololipidomics (see Methods). (A) Representative MRM chromatographs. (B) Representative MS-MS of MaR1 and RvD5. (C) MaR1 and total SPM levels (pg/ml; 0.5x106 cells); mean±SEM, n=5. No statistical significances were obtained among groups; one-way ANOVA with Tukey’s multiple comparisons test. Supplemental Figure 8 A

250000 U75302 100 nM

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veh 150000 Vehicle MaR1MaR [10 nM] MFI U75302 [100 nM] 100000 MaR1+MaR1+U75302U75302

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20 (above vehicle)

% increase of phagocytosis 10 ** 0

MaR1 MaR1 MaR1

MaR1+U75302 MaR1+U75302 MaR1+U75302

Non-transfected SC-shRNA LGR6-shRNA

Supplemental Figure 8. MaR1-stimulated phagocytosis with human macrophages is not altered by an BLT1 antagonist. Human MΦ (non-transfected, non-target siRNA or LGR6 siRNA-transfected) were plated onto chamber slides (0.1 x106 cells/well), incubated with a BLT1 antagonist (U75302; 100 nM) or vehicle for 10 min, followed by MaR1 (10 nM) or vehicle control for 15 min at 37°C. BacLight Green-labeled E. coli was then added to initiate phagocytosis. Fluorescent images were then recorded every 10 min for 60 min. Three separate experiments were carried out. In each experiment, 4 fields (40X) per condition (per well) were recorded. (A) Results are mean fluorescence intensity (MFI)/cell of 4 fields/condition from one representative experiment with non-transfected MΦ. (B) Results are % increase of phagocytosis above vehicle controls; mean±SEM, n=3. **P<0.01, vs MaR1 in non-transfected, SC-shRNA groups. No statistical significances were obtained between MaR1 and MaR1+U75302 in non-transfected, SC-shRNA and LGR6-shRNA groups; two-way ANOVA with Bonferroni’s multiple comparisons test. Supplemental Figure 9 A

150000 MaR1

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Supplemental Figure 9. R-spondin-2 reduces MaR1-stimulated phagocytosis with human macrophages. Human MΦ were plated onto chamber slides (0.1 x106 cells/well), incubated with R-spondin-2 (Rspo-2; 10 nM) or vehicle for 10 min, followed by MaR1 (10 nM) or vehicle control for 15 min at 37°C. BacLight Green-labeled E. coli was then added to initiate phagocytosis. Fluorescent images were then recorded every 10 min for 60 min. Three separate experiments were carried out. In each experiment, 4 fields (40X) per condition (per well) were recorded. (A) Results are mean fluorescence intensity (MFI)/cell of 4 fields/condition from one representative experiment. (B) Results are % increase of phagocytosis above vehicle controls; mean±SEM, n=3. *P<0.05, **P<0.01, vs MaR1 at 30 min; two-way ANOVA with Bonferroni’s multiple comparisons test. Supplemental Figure 10 A H A - H - - SSC SSC FSC FITC+ 61.4

GFP FSC-A SSC-W FSC-W

B Ren.713 shRNA LGR6.1 shRNA LGR6.2 shRNA LGR6.3 C 100 100 100 100 100 100

80 80 80 80 ** 60 60 60 60 50 50 *** *** 40 40 Normalized To Mode 40 Counts

Normalized To Mode 40 Normalized To Mode Normalized To Mode 20 20 20

20 (%) knockdown LGR6 LGR6 Knockdown (%)

0 00 0 0 3 4 0 0 10 10 3 4 3 4 0 10 10 0 10 10 3 4 Comp-PE-A 0 10 10 LGR6Comp-PE-A LGR6Comp-PE-A Comp-PE-A SGEP Empty shRNA LGR6.1shRNA LGR6.2shRNA LGR6.3 Ren.713 Control

Supplemental Figure 10. Stable LGR6 knockdown in HEK cells and MaR1 signaling with peripheral blood leukocytes Stable LGR6 knockdown in HEK293 cells. (A) Gating strategy for flow cytometry: HEK 293 cells were gated on FSC and SSC dot plots (left panel), then GFP positive (GPF+) populations were selected on the histograms (right panel). (B) LGR6 expression was determined within the GFP+ population. Results are representative histograms of Renilla 713 shRNA control and different clones of LGR6 reporter knockdown. (C) Each shRNA LGR6 reporter knockdown clone was calculated and compared to controls Renilla 713 shRNA control and empty mock SGEP vector. Results are mean±SEM; n=4. **p<0.01, ***P<0.001 vs. controls shRNA Renilla 713 or mock vector; one-way ANOVA with Tukey’s multiple comparisons test. Supplemental Figure 11

N-terminus

LGR6

7-TM MaR1

Gas b-arrestin

hLGR6 Recombinant hLGR6 hLGR6 mLGR6 mLGR6

Epithelial-like cells CHO-hLGR6 HEK-hLGR6 PMN ↓ Chemotaxis Macrophage ↑ b-arrestin recruitment ↑ CREB, ERK phosphorylation ↑ Cyclic AMP ↑ Cyclic AMP ↑ Impedance change ↑ [3H]-MaR1 binding ↑ [3H]-MaR1 binding ↑ Phagocytosis of E. coli, STZ ↑ Wound repair ↑ Efferocytosis ↑ CREB, ERK phosphorylation Monocytes

↑ Phagocytosis of E. coli ↑ CREB, ERK phosphorylation

Supplemental Figure 11. Illustration of MaR1-LGR6 dependent signaling and functions. Predicted LGR6 structure and MaR1 docking using UCSF Chimera (https://www.cgl.ucsf.edu/chimera/). LGR6 consists of N-terminus with leucine-rich repeats (light blue), and seven transmembrane domains (7-TM; dark blue). MaR1 3D structure is depicted in red, and predicted to interact with the transmembrane domains of LGR6. MaR1-LGR6 interactions initiate G coupling and response signals including impedance change, cAMP increase with recombinant LGR6. With phagocytes, MaR1-LGR6 interactions stimulate key pro-resolving functions with human and mouse MΦ, i.e., enhancing MΦ phagocytosis and efferocytosis. In addition, MaR1-LGR6 evoked phosphorylation signals with human and mouse monocytes and PMN, as well as limiting human PMN chemotaxis. Graphic Abstract

LGR6 N-terminus receptor

MaR1

7-TM

Gas b-arrestin

PMN Monocytes Monocytes PMN Macrophages Macrophages

Chemotaxis Phosphorylation Phagocytosis

pp38MAPK

pAKT

pERK1/2

pCREB

pS6

pNF-kBp65