doi:10.1038/nture09973 Plsm Memrne Phgosome TLR1/2/4 ROS Mitochondrion ROS OXPHOS Complex I ROS TRAF6 NADPH Oxidse Supplementry Figure 1 Model detiling the roles of mitochondril ROS in mcrophge cteril killing. Agonists derived from phgocytized cteri signl through TLR1/2, 2, or 4, leding to the recruitment of mitochondri to phgosomes nd concomitnt trfficking of TRAF6 to peri-phgosoml mitochondri where it intercts with nd uiquitintes. uiquitintion nd/or enrichment on the mitochondril outer memrne lters OXPHOS ctivity, resulting in incresed genertion of the mros superoxide. Superoxide is dismutted into hydrogen peroxide, which cn freely cross mitochondril memrnes to ugment ROSdependent killing of cteri. This cn occur s mros enters the phgosome nd directly contriutes to killing nd/or y mros priming of Phox signling components for full ROS genertion inside the phgosome. WWW.NATURE.COM/NATURE 1
Men coloclized pixels per ed Percent eds with mitochondril cupping Supplementry Figure 2 TLR1/2/4 signling induces mitochondril recruitment nd cupping round phgosomes. -, BMM were incuted with either uncoted, Pm3CSK4, or LPS coted 3 micron Fluoresrite ltex eds, fixed, nd mitochondril networks were immunostined with HSP70 ntiodies. Confocl Z-stcks were tken of three lrge fields of view, ech contining pproximtely 30 cells, nd coloclized imges were generted. Men coloclized pixels per ed () nd percent eds with mitochondril cupping () were clculted s descried in Methods. Error rs indicte s.d. from the men of triplicte imges. WWW.NATURE.COM/NATURE 2
Mito Mito Dpi Merge Merge c Lyste Nuc Cyt Mito d Immuno-gold leling (rrows indicte leling on outer mito mem) MAVS IκBα e Outer mem Intermem spce Inner mem Mtrix Whole mito MAVS (top nd) [Outer mem] f %TX-100 0.01 0.05 0.1 0.2 0.4 1 CytC [Intermem spce] COX1 [Inner mem] Hsp70 [Inner mem/mtrix] VDAC [Outer mem] GRIM19 [Inner mem] CytC [Intermem spce] solule pelleted mitos s p s p s p s p whole mitos Supplementry Figure 3 loclizes to oth mitochondril memrnes., NOR10 firolsts were immunostined with ntiodies ginst or mitochondril HSP60 [Mito]. Nuclei were stined with Dpi nd imges were collected nd merged., BMM were immunostined with ntiodies ginst or HSP60 [Mito] nd imges were collected nd merged. c, RAW cells were homogenized nd sujected to differentil centrifugtion nd sucrose grdient frctiontion. Frctions were lotted with the indicted ntiodies. d, J774 cells were stined with ntiody for immuno-electron microscopy nlysis. Arrows indicte gold prticles leling the outer memrne. e, RAW cell mitochondri were sufrctionted into outer memrne, intermemrne spce, inner memrne, nd mtrix comprtments or left intct nd lotted s indicted. f, RAW mitochondri were sujected to incresing concentrtions or 1% Triton X-100 to soluilize mitochondril memrnes or entire mitochondri, respectively. Smples were centrifuged to seprte soluilized proteins [s] from remining mitochondril pellets [p] nd lotted s indicted. WWW.NATURE.COM/NATURE 3
+Vector +-FLAG +HA-U +HA-U +MyD88 +TRAF6 +Vector +Vector +Vector +MyD88 +TRAF6 +Vector +ΔN--FLAG +-FLAG IB: HA IB: IP: FLAG - FLAG IP: FLAG IB: - FLAG -MG132 +MG132 IB: HA +TRAF6 +HA-U ΔN - FLAG c CTRL sh TRAF6 sh 0 15 30 60 120 240 0 15 30 60 240 120 min LPS IP: IB: U IB: IB: TRAF6 Lyste IB: IB: IκBα Supplementry Figure 4 is uiquitinted during TLR4 signling in TRAF6 dependent fshion. -, 293T cells were trnsfected with the indicted constructs, incuted in MG132 or not, lysed in oiling SDS uffer, nd immunoprecitted with FLAG ntiody. Smples with either proed with or HA ntiodies to detect uiquitin lddering. The ΔN--FLAG construct () lcks the N-terminl 260 residues required for TRAF6 interction. c, RAW cells stly expressing CTRL or TRAF6 shrna lentiviruses were stimulted with 1 μg/ml LPS for the indicted times nd lysed with oiling SDS uffer to denture proteins nd disrupt interctions. Smples were diluted in 2X TNT lysis uffer nd immunoprecipitted with ntiody. Whole cell lyste nd IP frctions were proed with the indicted ntiodies. WWW.NATURE.COM/NATURE 4
Unstimulted 30 LPS Gold prticles y EM 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Interior Periphery Unstimulted 30 LPS n=8 n=10 Supplementry Figure 5 ecomes more peripherl on mitochondri upon LPS stimultion. J774 cells were stimulted s indicted, fixed, sectioned, nd stined with ntiody for immuno-electron microscopy nlysis. For grph t ottom, gold prticles from EM imges of mitochondri (numer indicted elow columns) were quntified nd represented s percentge stining ner inner (interior) or outer (periphery) memrnes. WWW.NATURE.COM/NATURE 5
Inputs Cyto Mito Cyto Mito ++ - + - + + Mitos - ++ + + proteinse K - + sponin NDUFS3 HSP60 TRAF6 1 2 3 4 5 6 7 8 9 10 11 12 GFP sh 30 LPS TRAF6 sh 30 LPS GFP sh 30 LPS TRAF6 sh 30 LPS Supplementry Figure 6 TRAF6 is required for enrichment of t the mitochondril periphery upon TLR4 signling, ut does not enter mitochondri. GFP sh or TRAF6 sh RAW cells were stimulted with 1 μg/ml LPS nd mitochondri were frctionted. Equl mounts of extrcts were treted with the indicted mount of proteinse K (++, 12 ng/μl; +, 6 ng/μl) on ice with or without 0.1% sponin to gently permeilize mitochondril memrnes. Extrcts were then lotted with the indicted ntiodies. WWW.NATURE.COM/NATURE 6
20 min ed internliztion Pm3CSK4 LPS Pm3CSK4 LPS Pm3CSK4 LPS Mito Ltex eds Mito Colocliztion WT BMM TRAF6 KO BMM sh BMM Men coloclized pixels per ed Supplementry Figure 7 TRAF6 nd re required for mitochondril recruitment to phgosomes., BMM were incuted with either Pm3CSK4 or LPS coted Fluoresrite ltex eds, fixed, nd mitochondril networks were immunostined with HSP70 ntiodies [Mito]. Confocl Z-stcks were cquired nd coloclized eds (red pixels) nd mitochondri (green pixels) re displyed yellow (ottom). Imges shown re representtive of pproximtely 100 cells nlyzed., Confocl Z-stcks were tken of three lrge fields of view from oth Pm3CSK4 nd LPS coted ed smples, ech contining pproximtely 30 cells, nd coloclized imges were generted. Men coloclized pixels per ed were clculted s descried in Methods. Error rs indicte s.d. from the men of triplicte imges. WWW.NATURE.COM/NATURE 7
WT +/- VDAC BMM 6 hr stim c BMM 6 hr stim LPS LTA CpG LPS LTA CpG WT +/- mros Cellulr H2O2 unstined control ROS dye lone ROS dye + gonist Supplementry Figure 8 heterozygous BMM disply decresed mitochondril nd cellulr ROS upon TLR2/4 signling., WT or +/- littermte BMM were soluilized with TNT lysis uffer nd lotted using the indicted ntiodies. -c, WT or +/- littermte BMM were left untreted or stimulted with 1 μg/ml LPS, 2 μg/ml LTA, or 1 μm CpG DNA for 6 hours. Cells were stined with MitoSOX [mros] () or CM-H 2 DCFDA [cellulr H 2 O 2 ] (c) nd nlyzed y FACS. WWW.NATURE.COM/NATURE 8
LTA 24 hrs WT CTRL sh WT TRAF6 sh +/- sh Cellulr H2O2 mros unstined control ROS dye lone ROS dye + gonist Supplementry Figure 9 TRAF6- signling controls ROS genertion upon exposure to LTA. WT or +/- littermte BMM were infected with control, TRAF6, or shrna expressing lentiviruses nd left untreted or stimulted with 2 μg/ml LTA for the indicted time. Cells were stined with MitoSOX nd CM-H 2 DCFDA nd nlyzed y FACS. WWW.NATURE.COM/NATURE 9
100μM ntimycin A 2 hrs 0.5μM rotenone 16 hrs 10μM rotenone 5 hrs CTRL sh sh mros mros Supplementry Figure 10 knockdown cells generte norml levels of mros when exposed to ntimycin A or rotenone. -, RAW mcrophges stly expressing CTRL or shrnas were treted with ntimycin A () or rotenone () for the indicted times, stined with MitoSOX, nd nlyzed y FACS. WWW.NATURE.COM/NATURE 10
Fold increse over unstimulted Fold increse over unstimulted Fold increse over unstimulted Fold increse over unstimulted doi:10.1038/nture09973 GFP sh TRAF6 sh1 sh4 GFP sh TRAF6 sh1 sh4 TRAF6 VDAC WT +/- 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 WT BMM FACS men fluor. intensity - Cellulr H2O2 GFP sh TRAF6 sh1 sh4 2 6 24 hrs LPS stim 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 +/- BMM FACS men fluor. intensity - Cellulr H2O2 GFP sh TRAF6 sh1 sh4 2 6 24 hrs LPS stim c FACS men fluor. intensity - Cellulr H2O2 4.0 GFP sh 3.5 TRAF6 sh1 sh4 3.0 2.5 2.0 1.5 1.0 WT BMM +/- BMM FACS men fluor. intensity - Cellulr H2O2 4.0 GFP sh 3.5 TRAF6 sh1 sh4 3.0 2.5 2.0 1.5 1.0 0.5 2 6 24 hrs Pm3CSK4 stim 0.5 2 6 24 hrs Pm3CSK4 stim Supplementry Figure 11 TLR1/2/4 signling induces cellulr ROS through nd TRAF6., WT or +/- littermte BMM were infected with control GFP, TRAF6, or shrna expressing lentiviruses, soluilized in TNT lysis uffer, nd lotted using the indicted ntiodies. -c, WT or +/- littermte BMM were infected with GFP, TRAF6, or shrna expressing lentiviruses (unique shrna sequences; see Methods for more informtion) nd left untreted or stimulted with 500 ng/ml LPS () or 1 μg/ml Pm3CSK4 (c) for the indicted times. Cells were stined with CM-H 2 DCFDA nd nlyzed y FACS. Men fluorescence intensity fold chnges were clculted s descried in Methods. WWW.NATURE.COM/NATURE 11
Extrcellulr H2O2 (nm) doi:10.1038/nture09973 Het-killed Slmonell 5 hrs WT CTRL sh WT TRAF6 sh +/- sh Cellulr H2O2 mros unstined control ROS dye lone ROS dye + Slmonell 1400 1200 WT CTRL sh +/- sh 1000 800 600 400 200 0 3 hrs 6 hrs Live Slmonell MOI 5 Supplementry Figure 12 TRAF6 nd deficient BMM produce less mros nd cellulr H 2 O 2 upon exposure to Slmonell., WT or +/- littermte BMM were infected with CTRL, TRAF6, or shrna expressing lentiviruses nd left untreted or exposed to het-killed Slmonell t MOI of 50 cteri/cell for 5 hours. Cells were stined with MitoSOX nd CM-H 2 DCFDA nd nlyzed y FACS., WT or +/- littermte BMM were infected with CTRL or shrna expressing lentiviruses nd left untreted or stimulted with live, serum opsonized Slmonell t MOI of 5 cteri/cell for the indicted times. Extrcellulr H 2 O 2 production ws mesured y the Amplex Red Hydrogen Peroxide/Peroxidse ssy. Error rs represent s.d. of the men from triplicte smples. WWW.NATURE.COM/NATURE 12
Extrcellulr H2O2 (nm) doi:10.1038/nture09973 PMA 1 hr 4 hrs PMA 1 hr 4 hrs WT CTRL sh +/- sh Cellulr H2O2 mros unstined control ROS dye lone ROS dye + gonist c 250 200 WT CTRL sh +/- sh 150 100 50 0 100ng/ml PMA 30 min Supplementry Figure 13 knockdown cells generte norml levels of ROS when exposed to PMA. -c, WT or +/- littermte BMM were infected with CTRL or shrna expressing lentiviruses nd left untreted or stimulted with 100 ng/ml PMA for the indicted times. Cells were then stined with CM-H 2 DCFDA () or MitoSOX () nd nlyzed y FACS. Extrcellulr H 2 O 2 production ws mesured y the Amplex Red Hydrogen Peroxide/Peroxidse ssy (c). Error rs represent s.d. of the men from triplicte smples. WWW.NATURE.COM/NATURE 13
80 70 60 WT CTRL sh WT TRAF6 sh +/- sh Nitrite (µm) 50 40 30 20 10 0 Unstim 48 hrs LPS Cytokine Concentrtion (ng/ml) 10 9 8 7 6 5 4 3 2 1 0 WT CTRL sh WT TRAF6 sh +/- sh Unstim 24 hrs LPS Unstim 24 hrs LPS IL-12p40 TNFα Supplementry Figure 14 Nitric oxide nd proinflmmtory cytokine production re not inhiited in deficient BMM. -, WT or +/- BMM were infected with CTRL, TRAF6, or shrna expressing lentiviruses nd left untreted or stimulted with 500 ng/ml LPS for 48 hours. Superntnts were nlyzed for nitrite genertion y Griess regent () or IL-12p40 nd TNFα production y ELISA (). Error rs represent s.d. of the men from triplicte smples. WWW.NATURE.COM/NATURE 14
Cellulr H2O2 (fold increse over unstimulted) FACS men fluor. intensity 11.0 WT 9.0 MCAT 7.0 5.0 3.0 1.0 2 6 24 hrs LPS exposure Supplementry Figure 15 MCAT mcrophges generte less cellulr ROS when stimulted with LPS. WT nd MCAT littermte BMM were left untreted or stimulted with 500 ng/ml LPS for 2, 6, or 24 hours. Cells were then stined with CM-H 2 DCFDA nd nlyzed y FACS. Men fluorescence intensity fold chnges were clculted s descried in Methods, nd error rs represent s.d. of the men from triplicte smples. WWW.NATURE.COM/NATURE 15
Supplementry Tle 1 Mitochondril proteins identified in tndem ffinity purifiction from RAW 264.7 nd HEK293 cells. Entrez Gene ID Description RAW TAP Peptides 293 TAP Peptides 11740 solute crrier fmily 25 (mitochondril crrier, denine nucleotide trnsloctor), memer 5 5 9 15510 het shock protein 1, HSP60 5 10 229211 cyl-coenzyme A dehydrogense fmily, memer 9 12 9 298682 ATPse fmily, AAA domin contining 3A 1 6 498 ATP synthse, mitochondril F1 complex, lph suunit, isoform 1 0 11 68349 NADH dehydrogense (uiquinone) Fe-S protein 3 0 3 18674 solute crrier fmily 25 (mitochondril crrier, phosphte crrier), memer 3 69702 NADH dehydrogense (uiquinone) 1 lph sucomplex, ssemly fctor 0 3 6 0 WWW.NATURE.COM/NATURE 16