Supplementary Methods

1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 31 32 Supplementary Methods Gentamicin protection assay THP-1 monocytes were seeded onto a 12-well plate and differentiated into macrophages for 24 hours using 1 nm phorbol 12-myristate 13-acetate (PMA) in RPMI 164 (Gibco, Life Technologies Inc., USA). 9 minutes before infection cells were washed with PBS and an incomplete RPMI 164 medium was added onto cells. Cells were infected at a multiplicity of infection (MOI) of 5:1 using wild-type Salmonella Typhimurium (ATCC strain 1223) for 15 minutes, 9 minutes and 12 minutes. The cell culture supernatant was removed at each time point, and cells were washed twice with pre-warmed PBS. The cells were then supplemented with RPMI media 164 containing 1 µg/ml of gentamicin and incubated for one hour. The cell culture supernatant was removed, and cells were washed twice with PBS. Buffer containing.1% Triton X-1 in PBS was placed on cells for 15 minutes, after which serial dilutions were plated on agar plates and incubated at 37 C for 16 hours. Cells were counted, and cell viability was measured by using an automatic cell counter (Bio-Rad, USA) before and infection to calculate the CFU/cell. RT-PCR Total RNA was isolated from uninfected and S. Typhimurium-infected THP-1 cells (MOI 5:1, 1.5 hpi) by using the Bio-Rad Aurum Total RNA Mini Kit following the manufacturer s instructions. hour-control represents cells before the 1.5-hour incubation, 1.5 hour- control represents cells incubated for 1.5 hours in incomplete media, 1.5 hour Infected represents cells infected for 1.5 hours. Reverse transcription of 1 ng total RNA was completed using the iscripttm cdna Synthesis kit following manufacturer s instruction. The cdna reactions were incubated at 25 C for 5 minutes, 42 C for 3 minutes and 85 C for 5 minutes using the Bio-Rad CFX96 Real-Time System unit on the C1 Touch Thermal Cycler. The nucleic acids were analyzed using the BioTek Take3 plate and analyzed for absorbance at 26 nm and 28 nm to calculate the purity of the nucleic acids. All the A26/A28 ratio for RNA and cdna was ~2. and ~1.8, respectively. Quantitative PCR was completed with 4 ng of cdna, iq SYBR Green Supermix (Bio-Rad), OTUB1 and beta-actin forward and reverse primers (Bio-Rad) Prime PCR Assay. The optimized cycling protocol used for the complete qpcr samples were as following: initial denaturing was done at 95 C for 3 minutes, denaturing was done at 95 C for 15 seconds, and 1

33 34 35 36 37 38 39 4 41 42 43 44 45 46 47 48 49 5 51 52 53 54 55 56 57 58 59 6 61 62 63 64 annealing/extension was done at 55 C for 3 seconds. The denaturing and annealing/extension steps were completed for 4 cycles. OTUB1 transcripts (delta-delta Ct values) were quantified relative to beta-actin transcripts. Data (two technical replicates and three biological replicates) were analyzed in CFX Manager Software (v 3.1.1517.823, Bio-Rad) and p-values were calculated. Active-site probe labeling Human influenza hemagglutinin (HA)-tagged ubiquitin vinyl sulfone ubiquitin-specific active-site probe (Ub-VS-HA) was used to analyze the activities of deubiquitinating enzymes (DUBs). THP-1 macrophages were infected (or not) for, 3, 6 and 9 minutes with S. Typhimurium as described above. Cell culture supernatant was collected and cell debris removed by centrifugation (5 g for 1 minutes, followed by 9, x g for 1 min, where all steps were performed at 4 C). Sample containing equal amounts of protein were subjected to the reactions with Ub-VS-HA (Boston Biochem, USA) as we described previously (1, 2). The DUBs reacting with Ub-VS-HA probe were analyzed by SDS-PAGE, followed by Western blotting by using anti-ha antibody and OTUB1 antibody. Silver stain Silver stain was used to analyze SDS-PAGE containing CD63+ (F2) and CD9+ (F1) exosome samples from 4 µg of fractionated exosomes derived uninfected or Salmonella-infected RAW 264.7 macrophages and.1 µg LPS. The gel was fixed in 4% ethanol and 1% acetic acid for 1 hour. Next, the gel was washed with 3% ethanol for 2 minutes twice and subsequently washed with MilliQ water once for 2 minutes. For the silver reaction, the gel is then incubated in.2% sodium thiosulfate solution and then incubated with cold.1% Silver Nitrate solution containing 37% formalin for 2 minutes. After incubation, the gel is then washed in MiliQ water three times for 2 seconds each. The gel was developed solution containing 3% sodium carbonate with 37 % formalin and washed with miliq water for 2 seconds. The development of the gel is stopped by washing the gel in 5% acetic acid for 1 minutes and washed with MilliQ water for 5 minutes. 2

65 66 67 68 69 7 71 72 73 74 75 76 77 78 79 8 81 82 83 84 85 86 87 88 89 9 91 92 93 94 95 Cell Treatment with LPS and Polymyxin B RAW 264.7 macrophages were seeded in 24 well plates 24 hours before treatment. The following day the supernatant was aspirated and replaced with either DMEM complete media with or without 2 µg of polymyxin B for one hour before treatment with LPS or exosomes. LPS (5 pg or 1 pg) and exosomes (1 µg) were used to treat cells containing complete media with or without polymyxin B for 24 hours. The supernatant was collected and spun at 8 x g to remove cells and subsequently analyzed for TNF-α using ELISA. Cell Treatment of Exosomes with Proteinase K Raw 264.7 macrophages were seeded in 24 well plates 24 hours before treatment. Exosomes were lysed at 98 C for 1 minutes and then incubated in complete media containing 2 µg Proteinase K for 1 hour at 37 C. The enzymatic activity of proteinase K was inhibited by adding 1 µm of phenylmethylsulfonyl fluoride (PMSF). The lysed or intact exosomes incubated with or without proteinase K were used to treat cells for 24 hours. The supernatant was collected and spun at 8 g to remove cells and subsequently analyzed for TNF-α using ELISA. Limulus Amebocyte Lysate (LAL) Assay LAL assay was used to quantify endotoxin in crude or fractionated exosomes derived from uninfected of S. Typhimurium-infected macrophages. A 96 well microplate was equilibrated at 37 C for 1 minutes. Samples and standards were dispensed into the microplate and incubated at 37 C for 5 minutes. Next, LAL solution was added to each well, and the plate was placed on a plate shaker for 1 seconds then incubated at 37 C for 1 minutes. After that, a substrate solution is added to each well, mixed for 1 seconds using a plate shake and incubated at 37 C for 6 minutes. Finally, stop reagent (25% acetic acid) is added to each well and mixed for 1 seconds. The assay is read using the Cytation3 plate reader (Biotek, USA) at 45 nm. GW4869 Treatment of cells for ELISA assay RAW 267.4 macrophages were seeded on 5 mm dishes 24 hours before treatment. Macrophages were incubated with 5 µm of GW4869 or the vehicle control containing DMSO one hour before infection. Exosomes derived from uninfected or infected macrophages with GW4869 or DMSO were isolated and used to treat naïve macrophage for 24 hours. The 3

96 97 98 99 1 11 12 13 14 15 16 17 18 19 11 111 112 113 114 115 116 117 118 119 12 121 122 123 124 125 126 127 supernatant was collected and spun at 8 x g to remove cells and subsequently analyzed for TNF-α using ELISA. CFSE labeling of exosomes RAW 264.7 mouse macrophages were infected with wild-type S. Typhimurium (UK-1; MOI 5:1) or left uninfected. Exosomes were collected from cell culture supernatant two hpi. Exosomes were then stained with ExoGreen carboxyfluorescein succinimidyl diacetate ester (CFSE) dye (SBI), after which the exosomes were precipitated by using ExoQuick (SBI) reagent and spun down at 21, x g and the pellet was resuspended in PBS, and their concentration was established by using a BCA protein assay. Naïve RAW 264.7 macrophages were plated on 6-well plates and treated with PBS (Ctrl) or with.1 µg ExoGreen-labeled exosomes derived from infected and uninfected macrophages. After 2 hours the cells were examined by fluorescent microscopy by using a GFP channel and contrast phase (EVOS Cell Imaging Systems, Thermo Fisher Scientific), and both images were merged. The cells with internalized exosomes were labeled with ExoGreen dye. Confocal microscopy BMDMs were cultured on coverslips and treated for 24 hours with F1 exosomes isolated from infected or uninfected RAW 264.7 macrophages. Cells were fixed and stained with rhodaminephalloidin to visualize actin cytoskeleton. DAPI was used to stain nucleus, and differential interference contrast (DIC) was used to image cell morphology. Images were acquired by using Confocal Zeiss LSM8 microscope. Supplementary Tables Table S1. Extracellular proteins of human macrophages with abundance altered upon Salmonella enterica Typhimurium infection 4

128 129 Supplementary Figures 15 average CFU/cell 1 5 13 131 132 133 134 135 136 137 138 139 14 15 9 12 time [min] Figure S1. Gentamicin protection assay. THP-1 macrophages were infected at an MOI of 5:1 using wild-type Salmonella Typhimurium (ATCC strain 1223) for 15 minutes, 9 minutes and 12 minutes. The cell culture supernatant was removed at each time point, and cells were washed twice with pre-warmed PBS. The cells were then supplemented with RPMI media 164 containing 1 µg/ml of gentamicin and incubated for one hour. The cell culture supernatant was removed, and cells were washed twice with PB, lysed with.1% Triton X-1 and dilutions plated on LB plate for enumeration of CFUs. Viable THP-1 cells were counted to calculate the CFU/cell. 5

141 142 143 144 145 146 147 148 149 15 151 152 Figure S2. OTUB1 transcript analysis in infected cells. The OTUB1 transcript was quantified in uninfected and S. Typhimurium-infected THP-1 cells (MOI 5:1, 1.5 hpi) by using RT-PCR. hour-control represents cells before the 1.5-hour incubation, 1.5 hour- control represents cells incubated for 1.5 hours in incomplete media, 1.5 hour Infected represents cells infected for 1.5 hours. OTUB1 transcripts (delta-delta Ct values) were quantified relative to beta-actin transcripts. Data (two technical replicates and three biological replicates) were analyzed in CFX Manager Software (v 3.1.1517.823, Bio-Rad) and p-values were calculated. n.s., non-significant p-value. 6

153 154 155 156 157 158 159 16 161 162 163 Figure S3. Probing activity of extracellular deubiquitinating enzymes by HA-Ub-VS. THP-1 macrophages were infected or left uninfected for, 3, 6 and 9 minutes with S. Typhimurium. Cell culture medium was collected at each time point in duplicates. Extracellular proteins were exposed to the reaction with the ubiquitin-specific active-site probe (HA-Ub-VS), which reacts with the active site of deubiquitinating enzymes. Deubiquitinating enzymes that reacted with the probe were detected by anti-ha Western blotting. OTUB1 was also visualized by anti-otub1 western blotting. Protein content in cell pellet has been analyzed by using anti-otub1 and antiβ-actin western blotting. 7

A 8. B 7. Concentration (particles / ml) 6. 5. 4. 3. 2. 1. 7 121 BMDM Exo (-) Mean Size: 11.2 +/- 2.6 nm Mode SIze: 73.1 +/- 5.2 nm Concentration: 7.2 x 1^ 9 particle/ml Concentration [particles x 1^ 6/ml] BMDM Exo (+) Mean Size: 88.1 +/- 5.8 nm Mode Size: 58.2 +/- 2.1 nm Concentration: 7.4 x 1^ 9 particle/ml C 9. 241 346 481 1 2 3 4 5 6 7 8 9 1 Size (nm) D 8. Concentration (particles / ml) 7. 6. 5. 4. 3. 2. 92 123 168 CD63 Exo (-) Mean Size: 127.6 +/- 3.5 nm Mode Size: 121.4 +/- 2.3 nm Concentration: 7.54x 1^ 9 particle/ml CD9 Exo (-) Mean Size: 112.8 +/- 2.2 nm Mode Size: 84.5 +/- 1.6 nm Concentration: 1.26 x 1^ 9 particle/ml 1. E 1.4 241 1 2 3 4 5 6 7 8 9 1 Size (nm) F 1.4 129 1.2 Concentration (particles / ml) 1..8.6.4 51 84 125 CD63 Exo (+) Mean Size: 14.7 +/- 7.7 nm Mode Size: 96. +/- 15.2 nm Concentration: 1.4x 1^ 1 particle/ml Concentration (particles / ml) 1.2 1..8.6.4 173 189 CD9 Exo (+) Mean Size: 178.6 +/- 2.6 nm Mode Size: 129.3 +/- 2.2 nm Concentration: 2.8 x 1^ 1 particle/ml.2.2 281 164 165 166 167 168 169 17 171 172 173 263 345 455 571 1 2 3 4 5 6 7 8 9 1 Size (nm) Figure S4. NanoTracking Analysis (NTA) of exosomes. NTA was used to determine the mean, mode and concentrations of exosomes derived from uninfected (A) and S. Typhimurium- infected (B) BMDMs. Exosomes derived from infected [(D) and (F)] and uninfected [(C) and (E)] THP-1 cells were fractionated by density gradient and NTA analysis of CD63-enriched [(C) and (E)] as well as CD9-enriched [(D) and (F)] was performed. In each case, exosome samples were diluted until ~1^8 particles/ml were detected by Nanosight. Dilution factor was calculated, and the concentration was reported on each graph. Exo(+), exosomes derived from infected macrophages; Exo(-), exosomes derived from uninfected macrophages. 387 1 2 3 4 5 6 7 8 9 1 Size (nm) 536 8

174 175 176 177 178 179 18 181 182 183 184 185 186 187 188 Figure S5. RAW 264.7-derived exosomes are internalized by naïve RAW 264.7 cells. RAW 264.7 mouse macrophages were infected with wild-type S. Typhimurium (UK-1; MOI 5:1) or left uninfected. Exosomes were collected from cell culture supernatant two hpi. Exosomes were then stained with ExoGreen carboxyfluorescein succinimidyl diacetate ester (CFSE) dye (SBI), after which the exosomes were precipitated by using ExoQuick (SBI) reagent and spun down at 21, x g and the pellet was resuspended in PBS, and their concentration was established by using a BCA protein assay. Naïve RAW 264.7 macrophages were plated on 6-well plates and treated with PBS (Ctrl) or with.1 µg ExoGreen-labeled exosomes derived from infected and uninfected macrophages. After 2 hours the cells were examined by fluorescent microscopy by using a GFP channel and contrast phase (EVOS Cell Imaging Systems, Thermo Fisher Scientific), and both images were merged. The cells with internalized exosomes were labeled with ExoGreen dye (as an example are shown RAW 264.7 macrophages treated with exosomes derived from infected 264.7 cells), but none of the cells treated with ExoGreen dye alone were labeled under these conditions (not shown). 189 19 191 9

TNF- α [pg/ml] 15 1 5 **** **** ** VC GW4869 VC + Inf GW4869 + Inf 192 193 194 195 196 197 198 199 Figure S6. TNF-α in macrophages treated with neutral sphingomyelinase inhibitor (GW4869) infected with S. Typhimurium. RAW 264.7 macrophages were treated with GW4869 [5 µm] or an equal volume of DMSO, a vehicle control (VC), and subjected to infection with S. Typhimurium for 2 hours (Inf) or left uninfected. The cell culture supernatant was collected and analyzed for TNF-α. One-way ANOVA with Tukey s test was used to test for statistical significance. P-values were indicated as follows: * p.5; ** p.1; *** p.1; **** p.1. 2 1

21 22 23 24 25 26 27 28 29 21 A B Relative abundance Relative abundance 8 DCs Exo 6 4 2 6 4 2 Figure S7. Exosomes stimulate chemokine release in naïve macrophages and DCs. (A). Exosomes produced from S. Typhimurium-infected [2 hpi; Exo (+)] and not infected [Exo (-)] murine bone marrow-derived DC (BMDCs) were used to treat naïve BMDCs for 24 hours. After 24 hours CCS was collected, and 4 chemokines were analyzed by Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D Systems, USA). The pixel intensity of spots was measured by ImageJ, the relative abundance was adjusted to the background and visualized as a graph. (B). CD63-positive exosomes (fraction F2) isolated from S. Typhimurium-infected (2 hpi) RAW 264.7 macrophages were used to treat naïve RAW 264.7 macrophages. Released chemokines were measured as in (A). sicam-1 IL-1ra CXCL1/CRG-2 CXCL1 8 F2 Exo CCL2/MCP-1 MIP-1α MIP-1ß MIP-2 RANTES SDF-1 TNF-α sicam-1 IL-1ra IP-1 MCP-1 MIP-1α MIP-1ß MIP-2 RANTES SDF-1 TNF-α Exo (+) Exo (-) Exo (+) Exo (-) 11

211 212 213 214 215 216 217 218 219 22 221 222 223 224 225 A TNF- α [pg/ml] 4 3 2 1 Ctrl Ctrl + PMB C **** LPS 5pg Endotoxin Units (EU)/ml LPS 5pg + PMB 4 3 2 1 **** **** Exo (-) n=3 **** LPS 1pg LPS 1pg + PMB Exo (+) **** Exo (+) HI Exo * Exo + PMB D Figure S8. Endotoxin is a cargo of exosomes. (A) RAW 264.7 macrophages were treated with LPS (5 pg/ml and 1 pg/ml) or exosomes (1 µg) derived from RAW 264.7 macrophages infected with S. Typhimurium (2 hpi) in presence or absence of 2 µg/ml of polymyxin B (PMB) for 24 hours. ELISA was used to quantify TNF-α release to cell culture supernatant. (B) Exosomes (1 µg) derived from Salmonella-infected macrophages were lysed or left intact and treated with or without Proteinase K (2 µg). RAW 264.7 cells were treated with equal amount of material for 24 hours, and ELISA was used to quantify release TNF-α. (C). LAL assay was used to quantify endotoxin in intact exosome preparations derived from uninfected macrophages as well as from intact and lysed (HI) exosomes derived from Salmonella-infected macrophages. Samples were analyzed in duplicate and a representative experiment is shown. (D). Protein and LPS components in F2 and F1 exosomes derived from uninfected and Salmonella-infected macrophages were analysed by SDS-PAGE and silver stain. As a control.1 µg LPS (S. Typhimurium) was used. Exo(+), exosomes derived from infected macrophages; Exo(-), exosomes derived from uninfected macrophages. One-way ANOVA with Tukey s test was used B TNF- α [pg/ml] 25 15 1 75 5 37 25 2 15 1 8 6 4 2 LPS Ctrl * Exo (+) n=4 * Exo (+) ProtK ** * Exo (+) HI F2(-) F2(+) F1(-) F1(+) Exo (+) HI ProtK 12

226 227 to test for statistical significance for all figures. P-values were indicated as follows: * p.5; ** p.1; *** p.1; **** p.1. 228 229 23 231 232 233 234 Figure S9. Subpopulations of THP-1 macrophage-derived exosomes from S. Typhimuriuminfected cells trigger TNF-α and IL-1β release in uninfected THP-1 macrophages. PBS control (Ctrl) or.1 µg exosomes isolated from S. Typhimurium-infected (2 hpi) THP-1 macrophages (fractions F2, F7, and F1) were used to treat naïve THP-1 macrophages for 24 hours, after which released IL-1 (A), IL-1β (B) and TNF-α (C) were quantified by ELISA assays. Exo(+), exosomes derived from infected macrophages; Exo(-), exosomes derived from uninfected 13

235 236 macrophages. P values were indicated as follows: * p.5; ** p.1; *** p.1; **** p.1. 237 238 F RhodaminePhalloidin DIC DAPI Merged Exo (-) F1 1 um 1 um 1 um 1 um Exo (+) F1 239 24 241 242 243 1 um 1 um 1 um 1 um Figure S1. Murine BMDMs were cultured on coverslips and treated for 24 hours with F1 exosomes isolated from S. Typhimurium-infected (2 hpi) or uninfected RAW 264.7 macrophages (B). Cells were fixed and stained with rhodamine-phalloidin to visualize actin cytoskeleton. DAPI was used to stain nucleus, and differential interference contrast (DIC) was used to image cells. 244 245 246 247 248 249 25 251 252 253 14

TNF- α [pg/ml] 1 8 6 4 * ** Ctrl Exo (-) Exo (+) 2 254 255 256 257 258 259 26 261 Figure S11. TLR 4-/- C57BL/6 macrophages were treated with exosomes derived from uninfected or infected (MOI 5:1, 2 hpi) RAW 264.7 macrophages or with PBS (control). After 24 hours of treatment concentration of TNF-α in CCS was measured by ELISA. Four biological replicates are shown. One-way ANOVA test with Tukey s multiple testing correction was used to establish statistical significance. P values were indicated as follows: * p.5; ** p.1; *** p.1; **** p.1. 262 263 264 265 266 267 268 269 27 271 272 273 274 15

275 276 277 278 279 28 281 282 283 284 Supplemental Bibliography: 1. Edelmann MJ, Kramer HB, Altun M, Kessler BM. 21. Post-translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA levels and susceptibility to Yersinia invasion. FEBS J 277:2515-3. 2. Kummari E, Alugubelly N, Hsu CY, Dong B, Nanduri B, Edelmann MJ. 215. Activity-Based Proteomic Profiling of Deubiquitinating Enzymes in Salmonella- Infected Macrophages Leads to Identification of Putative Function of UCH-L5 in Inflammasome Regulation. PLoS One 1:e135531. 16