1 SUPPLEMENARY INFORMAION 13 supplementry figures, 2 supplementry tles, nd supplementry note he leukocyte integrin ntgonist Del-1 inhiits IL-17 medited inflmmtory one loss Mehmet A. Eskn 1,2, Rvi Jotwni 2, oshihru Ae 2,9, Jindrich Chmelr 3, Jong-Hyung Lim 3, Shung Ling 2, Pul A. Ciero 2, Jennifer L. Kruss 2, Fenge Li 2, Mrtin Runer 4, Lorenz C. Hofuer 4, Eun Young Choi 3,5,6, Kyoung-Jin Chung 3, Ahmed Hshim 7, Michel A. Curtis 7, rintfyllos Chvkis 3,6,8, nd George Hjishengllis 1,2,8,9 1 Deprtment of Microiology nd Immunology, University of Louisville School of Medicine, Louisville, KY 4292, USA, 2 Center for Orl Helth nd Systemic Disese, University of Louisville School of Dentistry, Louisville, KY 4292, USA, 3 Division of Vsculr Inflmmtion, Dietes nd Kidney, Deprtment of Medicine nd Institute of Physiology, echnicl University Dresden, 137 Dresden, Germny, 4 Division of Endocrinology, Dietes, nd Bone Diseses, Deprtment of Medicine, echnicl University Dresden, 137 Dresden, Germny, 5 Deprtment of Medicine, Grdute School, University of Ulsn, Seoul 138-736, Repulic of Kore, 6 Experimentl Immunology Brnch, NCI, NIH, Bethesd, MD, 2892, USA, nd 7 Centre for Immunology nd Infectious Disese, Blizrd Institute, Brts nd he London School of Medicine nd Dentistry, Queen Mry University of London E1 2A, UK. 8 hese uthors contriuted eqully to this work. 9 Present ddress: Deprtment of Microiology, University of Pennsylvni, School of Dentl Medicine, 24 South 4th Street, Phildelphi, PA 1914, USA. Correspondence should e ddressed to G.H. (geoh@upenn.edu)
2 Del-1 expression (mrna copies / 1 3 GAPDH copies) 2 15 1 3 2 1 Brin Liver Gingiv Del-1 CD31 merge c d β-glctosidse CD31 merge Supplementry figure 1. Del-1 is expressed in the mouse gingiv y endothelil cells. () Brins, livers, nd gingiv were hrvested from 8-week-old C57BL/6 mice nd processed for quntittive rel-time PCR (qpcr) to determine Del-1 mrna expression (normlized ginst GAPDH mrna; dt re mens ± SD [n = 3 mice per group] from one of two independent experiments tht yielded similr results. Brin nd liver served s positive nd negtive control, respectively, for gingiv. () Sgittl sections of interdentl gingiv were stined for Del-1 nd CD31 (endothelil cell mrker), s indicted, with colocliztion (rrows) shown in the merged imge (scle r, 5µm). he lower row contins the overlys of the sme fluorescent confocl imges with corresponding DIC imges. (c) X-gl stining of gingiv from Edil3 -/- mice (left pnel); these mice re Del-1 knock-out/lcz knock-in trnsgenics where the LcZ gene is controlled y the ntive Del-1 promoter nd is thus reporter for Del-1 expression. he reltively restricted pttern of X-gl-stining in the connective tissue is contrsted with Del-1 stining in the confocl imge (middle pnel) involving oth the connective tissue nd the
epithelium (unlike Del-1, LcZ-encoded β-glctosidse is not secreted). As expected, no positive X-gl stining ws detected in wild-type control mice (right pnel). (d) Gingiv were stined for β-glctosidse nd CD31, s indicted, with colocliztion shown in the merged imge; shown re overlys of DIC nd fluorescent confocl imges (scle r, 5µm). he similr expression pttern of β-glctosidse (reporter for locl Del-1 expression) nd CD31 (endothelil cell mrker) indictes tht Del-1 is expressed y endothelil cells in the mouse gingiv. he immunohistochemicl nlysis is representtive of 5 () or 6 (c-d) mice. 3
4 G W Edil3 -/- G W Edil3 -/- B B G G B B Figure S4 B B B c e otl BMD (mg/cm³) Fluorescence intensity (% W control) 5 4 3 2 1 7 6 5 4 3 2 1 W Edil3 -/- reculr BMD (mg/cm³) d No. osteocltsts (RAP + cells) 2 1 8 6 4 2 W Edil3 -/- otl BMD (mg/cm³) 5 4 3 2 1 B reculr BMD (mg/cm³) 3 2 1 B W Edil3 -/- Distl femur Fourth lumr verter Supplementry figure 2. Del-1 deficiency is ssocited with incresed osteoclstic ctivity in the periodontium. () Sgittl sections of mxillry teeth from 2-week-old wild-type (W) or Edil3 -/- mice were stined for RANKL (scle r, 5µm;, tooth; G, gingiv; B, one). Shown re representtive overlys of DIC nd fluorescent confocl imges (upper pnels). here is incresed RANKL stining in regions djcent to the periodontl one in Edil3 -/- mice reltive to wild-type controls. In the lower pnels, the imges were lso stined with DAPI to visulize cell nuclei. Imges re representtive of 5 mice () Representtive histologicl sections (n = 5 mice) showing incresed numers of RAP-positive cells (rrows) in the periodontl one of 2-weekold Edil3 -/- mice s compred to wild-type controls (originl mgnifictions 2 & 4 in the upper nd lower pnels, respectively). (c) he RANKL fluorescence intensities of imges shown in A, nd of dditionl representtive imges from independent mice (5 per group) were quntified using ImgeJ nlysis. (d) Men numer (± SD) of RAP-positive cells determined in 1 rndom microscopic fields, including those shown in B (originl mgnifiction 2). (e) otl nd treculr one minerl density (BMD) ws mesured t the distl femur or the fourth lumr vertere of W or Edil3 -/- mice y peripherl quntittive computer tomogrphy. Numericl dt represent mens (± SD; n = 5-6 mice per group). P <.5; P <.1 compred to corresponding control.
5 mrna expression (reltive) 5 4 3 2 1 8 IL-17A IL-17F G-CSF NF IL-6 CXCL1 CXCL2 CXCL3 CXCL5 CCL2 CCL3 CCL2 W CXCR2 CCR1 CCR2 CCR6 C3R C5R REM-1 LR2 LR4 Edil3 -/- LR9 RANKL OPG cytokines chemokines & receptors innte receptors one W Edil3 -/- mrna expression (reltive) 6 4 2 IL-17A IL-17F G-CSF NF IL-6 CXCL1 CXCL2 CXCL3 CXCL5 CCL2 CCL3 CCL2 CXCR2 CCR1 CCR2 CCR6 C3R C5R REM-1 LR2 LR4 LR9 RANKL OPG cytokines chemokines & receptors innte receptors one Supplementry figure 3. Incresed mrna expression of inflmmtory meditors in the periodontium of Edil3 -/- mice. Gingiv were dissected from wild-type (W) or Edil3 -/- mice t the ge of 8 weeks () or 9 months () nd gingivl mrna expression of the indicted molecules ws determined y qpcr (normlized ginst GAPDH mrna nd expressed s fold chnge in Edil3 -/- trnscript undnce reltive to tht of W, the verge vlue of which ws tken s 1). Dt re mens ± SD (n = 4 mice per group). P <.5; P <.1 compred to corresponding W control.
6 Orl neroic cteri CFU (x1 4 ) c cteril numers (log 1 ) 12.5 1. 7.5 5. 2.5. 6 5 4 3 2 1 W Edil3 -/- 4 wk 5 wk 8 wk 16 wk 9 mo 8 W 7 Edil3 -/- 5 wk 16 wk 9 mo Chnge in one (mm). -.5-1. -1.5 untreted ntiiotics 14 wk 2 wk Supplementry figure 4. Edil3 -/- mice hror higher numers of orl cteri which re required for periodontl one loss. () Wild-type (W) nd Edil3 -/- mice t the indicted ges were ssessed for orl neroic cteri y culture; CFU re shown for ech mouse with horizontl lines denoting men vlues. () otl cteri were enumerted in the periodontl tissue of W nd Edil3 -/- mice t the indicted ges y quntittive rel-time PCR of the 16S rrna gene. Dt re mens ± SD (n = 5 mice per group). (c) Edil3 -/- mice were given ntiiotics (sulfmethoxzole nd trimethoprim) in the drinking wter since wening (plin wter served s control; untreted ) nd fter 14 or 2 weeks were ssessed for one loss (reltive to seline determined y 5-week-old Edil3 -/- mice ) Dt re mens ± SD (n = 5 mice per group). P <.5; P <.1 compred to corresponding W control or indicted group. At 5 weeks, Edil3 -/- mice hve not yet strted experiencing one loss nd their one heights re similr to those of W littermte controls (see Figure 2f).
7 c mrna expression (reltive) MPO (ng/mg totl protein) 3 2 1 2 1 unstimulted stimulted IL-17A NF 8 weeks 2 weeks pg/ml unstimulted 2 stimulted 15 2 1 IL-17A NF Supplementry figure 5. Mouse neutrophils express IL-17A nd their recruitment in the gingiv increses with dvncing ge. (-) Mouse neutrophils isolted from the one mrrow were treted with medium only (unstimulted) or stimulted with PMA (2 ng/ml) for 4h () or with PMA (2 ng/ml) plus ionomycin (1 µg/ml) for 4h () nd ssessed for IL-17A mrna expression or IL-17A protein relese, respectively (NF determintions served s positive controls). he mrna expression dt re from one representtive experiment (out of four independent experiments yielding similr results). he protein dt re mens ± SD (n = 3 sets of neutrophils per group) from one of three independent experiments tht yielded similr results. (c) Dissected gingiv from Edil3 -/- mice, of the indicted ges, were processed for ELISA determintion of MPO protein mounts. MPO served s mrker of neutrophil infiltrtion. Dt re mens ± SD (n = 6 mice per group). P <.1 compred to 8-week-old group.
8 IL-17 CD4 merge S S G W Edil3 -/- c Fluorescence intensity (% W control) 125 1 75 5 25 W Edil3 -/- d mrna expression (reltive) 7 6 5 4 3 2 1 W Edil3 -/- G PGRP-1 CD4 e W Edil3 -/- IL-17 γδ-cr merge IL-17 γδ-cr merge f Fluorescence intensity (% W control) 15 125 1 75 5 25 W Edil3 -/- Supplementry figure 6. IL-17 expression in Edil3 -/- gingiv y CD4 + or γδ-cr + cells. () Sections of interdentl gingiv from Edil3 -/- mice were stined for IL-17A nd CD4, s indicted, with colocliztion (rrows) shown in merged imges (scle r, 2 µm). () Sgittl sections of interdentl gingiv from 2-week-old wild-type (W) or Edil3 -/- mice were stined for CD4; shown re representtive overlys of DIC nd fluorescent confocl imges (scle r, 5µm;, tooth; G, gingiv; S, sulcus). (c) he fluorescence intensities of the imges shown in nd of dditionl representtive imges from independent mice were quntified using ImgeJ
nlysis. (d) Gingiv were dissected from W or Edil3 -/- mice nd gingivl mrna undnce of CD4 nd PGRP-1 (neutrophil mrker) ws determined y qpcr (normlized ginst GAPDH mrna nd expressed s fold chnge in Edil3 -/- trnscript undnce reltive to wild-type trnscript undnce, which ws ssigned n verge vlue of 1). (e) Sgittl sections of interdentl gingiv from 2-week-old W or Edil3 -/- mice were stined for IL-17A nd γδ CR, s indicted, with colocliztion (yellow) shown in merged imges (scle r, 2 µm). (f) he fluorescence intensities of the imges shown in e nd of dditionl representtive imges from independent mice were quntified using ImgeJ nlysis. Numericl dt re mens ± SD (n = 5 to 6 mice per group)., P <.1. he immunohistochemicl nlysis (-c & e-f) is representtive of 5 independent mice per group nd the qpcr experiment (d) ws repeted twice yielding similr results. 9 Note: he incresed expression of the neutrophil mrker PGRP-1 (d) correltes with other findings in the min pper showing incresed numers of neutrophils (confocl microscopy) nd elevted MPO mounts (ELISA), wheres the comprle CD4 expression in W nd Edil3 -/- mice (d) is consistent with the findings from the confocl microscopy nlysis (c).
1 W Edil3 - /- Edil3 - /- Il17r - /- Edil3 - /- Itgl - /- Supplementry figure 7. Mice with comined Del-1/IL-17R (Edil3 -/- Il17r -/- ) or Del-1 LFA- 1 (Edil3 -/- Itgl -/- ) deficiency re protected from periodontl one loss. Representtive imges of mxille (upper jws) showing tht the one gp (CEJ-ABC distnce; see crtoon) is gretest in 2-week-old mice with single Del-1 deficiency, wheres ge-mtched mice with comined Del-1 deficiencies hve one heights comprle to those of wild-type (W) mice. he imges re representtive of the mice used in the experiment shown in Figure 4 of the min pper.
11 Chnge in one (mm).2. -.2 -.4 -.6 -.8 W Il17r -/- 5-wk-old W Il17r -/- 2-wk-old Chnge in one (mm). -.1 -.2 -.3 -.6 -.8-1. W Edil3 -/- Edil3 -/- Il17r -/- Il17r -/- Supplementry figure 8. Periodontl one heights in Il17r -/- mice reltive to ge-mtched wild-type controls. () C57BL/6 Il17r -/- mice nd ge-mtched wild-type (W) controls were ssessed for periodontl one heights. Negtive vlues denote one loss reltive to the one heights of 5-week-old W mice (zero seline); the dotted line indictes the one heights of 2- week-old W mice. () C57BL/6 W mice or mice geneticlly deficient in the indicted molecules were ssessed for periodontl one heights t 3 weeks of ge. Negtive vlues denote one loss reltive to the one heights of 5-week-old W mice (zero seline); the dotted line indictes the one heights of 3-week-old W mice for ese of comprison with ge-mtched gene knockout mice. Dt re mens ± SD (, n = 7-1 mice per group;, n = 5-6 mice per group) from one of two independent sets of experiments tht yielded similr results., P <.5;, P <.1 compred to ge-mtched W littermte control.
12 MPO (% of unligted control) 14 12 1 8 6 4 2 Unligted (seline control) Ligted + PBS Ligted + Meloxicm Orl neroic cteri CFU 1 6 Unligted (seline control) Ligted + PBS 1 7 1 5 Ligted + Meloxicm 1 4 1 3 Supplementry figure 9. Inflmmtory nd microiologicl chnges in the periodontium fter nti-inflmmtory tretment. o induce periodontl inflmmtion, silk ligtures were tied round the second molr teeth of 1-week-old wild-type mice. One such group ws treted dily with meloxicm (1 mg/kg; s.c), nonsteroidl nti-inflmmtory drug tht selectively inhiits COX-2, nd nother group received PBS control. A third group ws not ligted or received tretment nd served s seline control. After 5 dys, the mice were scrificed nd ssessed for MPO mounts in dissected gingiv () nd for numers of orl neroic cteri (). In, dt re mens ± SD (n = 5 mice per group) In, CFU re shown for ech individul mouse with horizontl lines denoting men vlues. P <.1 etween the indicted groups. he dt re from one of two independent experiments tht yielded consistent results. Note: hese dt indicte tht nti-inflmmtory tretment cn reduce the orl cteril lod even though it lso reduced neutrophil infiltrtion, s indicted y diminished gingivl MPO mounts.
13 Del-1 expression (reltive) 1 1.1.1 helthy P =.41 disesed IL-17A expression (reltive) 1 P =.284 1 1.1.1 helthy disesed Supplementry figure 1. Expression of Del-1 nd IL-17A in gingivl iopsy smples from periodontitis ptients. Gingivl Del-1 () nd IL-17A () mrna expression in helthy nd disesed sites were determined y qpcr (normlized ginst GAPDH mrna nd expressed s fold chnge reltive to helthy-site trnscript undnce, which ws ssigned n verge vlue of 1). he pir of dt for ech individul is connected y line (n = 7 individuls). See Supplementl Methods for selection of ptients nd helthy plus disesed gingivl sites. Note: Helthy sites expressed on the verge 6.5-fold higher Del-1 mrna nd 17.9-fold lower IL-17A thn disesed (inflmed) sites. Both differences were sttisticlly significnt (P <.5). hese dt re consistent with findings in the mouse model (see min pper) tht Del-1 nd IL- 17A expressions re inversely ssocited in the periodontium.
14 mrna expression (reltive) 2.5 2. 1.5 1..5. IL-17A 5 weeks W Il17r -/- PGRP-1 mrna expression (reltive) 2.5 2. 1.5 1..5. IL-17A 8 weeks W Il17r -/- PGRP-1 MPO (ng/mg totl protein) 25 2 15 1 5 5 weeks 8 weeks W Il17r -/- Supplementry figure 11. Incresed IL-17A expression nd reduced neutrophil infiltrtion in the gingiv of Il17r -/- mice. Dissected gingiv from C57BL/6 wild-type (W) or Il17r -/- mice, of the indicted ges, were processed for qpcr determintion of IL-17A nd PGRP1 trnscript undnce () or ELISA determintion of MPO protein mounts (). he mrna expression ws normlized ginst GAPDH mrna nd expressed s fold chnge in Il17r -/- trnscript undnce reltive to wild-type trnscript undnce, which ws ssigned n verge vlue of 1. PGRP1 nd MPO oth served s mrkers of neutrophil infiltrtion. Dt re mens ± SD (n = 5-6 mice per group). P <.1 compred to corresponding W control.
15 MPO (ng/mg totl protein) 1 75 5 25 Unligted Ligted mrna expression (reltive) 32 Unligted Ligted 16 8 4 2 1.5.25.125 IL-17A IL-17F G-CSF CXCL1 CXCL2 CXCL3 CXCR2 IL-6 NF IL-1β MMP9 COX-2 RANKL OPG Del-1 Supplementry figure 12. Inflmmtory host responses in ligture-induced periodontitis. Ligture-induced periodontl inflmmtion in C57BL/6 mice ws monitored in dissected gingiv processed for determining MPO mounts y ELISA () or for qpcr to determine mrna expression of the indicted molecules, normlized ginst GAPDH mrna (). he dt re expressed s fold chnge in the trnscript undnce in the ligted side reltive to tht of the unligted side, which ws ssigned n verge vlue of 1. Dt re mens ± SD (n = 5-6 mice per group) from one of three independent experiments yielding similr results. P <.1 compred to corresponding control.
16 Migrtion (% of control) 1 c 5 W Edil3 -/- no chemokine CXCL2 NF (pg/ml) 5 W Edil3 -/- 4 3 2 1 unstimulted stimulted CXCL1 (pg/ml) 75 5 25 W Edil3 -/- unstimulted stimulted NF (pg/ml) 75 No Del-1 + Del-1 5 25 CXCL1 (pg/ml) 75 No Del-1 + Del-1 5 25 unstimulted stimulted unstimulted stimulted Supplementry figure 13. Effects of Del-1 deficiency or Del-1 on neutrophil function. () Migrtion of neutrophils isolted from wild-type (W) or Edil3 -/- mice ws tested in trnswell system. Chemokinesis (in the sence of chemokine in the ottom well) or chemotxis to CXCL2 (1 ng/ml) in the ottom well ws ssessed fter 6 min. he migrtion of W neutrophils in the sence of chemokine ws set s the 1% control (dshed line) nd the dt were expressed reltive to this vlue. () Neutrophils isolted from W or Edil3 -/- mice were stimulted or not for 4h with LPS (1 ng/ml) plus C5 (1 nm) nd relese of the indicted cytokines or chemokines in the culture superntnts ws determined y ELISA. (c) W neutrophils, in the presence or sence of solule Del-1 (1 µg/ml), were stimulted s ove nd ssyed for relese of the indicted cytokines or chemokines. Dt re mens ± SD (n = 3 sets of neutrophils), from one of two or three independent experiments yielding similr results. Notes: (-) No significnt differences were found in the cpcities of neutrophils from W or Edil3 -/- mice for migrtion or induction of NF or CXCL1 relese. (c) No significnt differences were oserved in the relese of NF or CXCL1 y neutrophils upon Del-1 tretment.
Supplementry tle 1. Quntifiction of colocliztion of vrious cell types with IL-17 in the gingivl tissue of Edil3 -/- mice. 17 Cell type (mrker used) γδ cells (γδ-cr) CD4 + cells (CD4) Neutrophils (Ly6G) % Colocliztion 8-wk-old mice 2-wk-old mice 68.4 ± 12.1 c 8.5 ± 9.7 d 23.3 ± 8.2 34.2 ± 1.6 17.8 ± 7.7 65.2 ± 14.9 d,e Colocliztion ws quntified y mens of ImgeJ intensity correltion nlysis of confocl sections of interdentl gingiv, similr to those shown in figure 4f nd supplementry figures 6 nd 6e. Mens ± SD (representtive confocl imges from independent mice; n = 5 mice per group). c Significntly (P <.1) higher compred to CD4 + cells nd neutrophils t 8 weeks. d Significntly (P <.1) higher compred to CD4 + cells t 2 weeks. e Significntly (P <.1) higher compred to corresponding 8-wk-old group (no sttisticlly significnt differences etween the two ge groups of CD4+ cells or γδ cells).
Supplementry tle 2. Reduced mrna expression of inflmmtory meditors in the gingiv of ged mice fter locl dministrtion of solule Del-1. 18 Fold chnge vs. Molecule Del-1 untreted controls CMC Pg IL-17.19.25 NF.5.51 IL-6.25.24 IL-1.5.25 RANKL.49.25 G-CSF.12.25 CCL2.48.49 CCL3 1.2.51 CCL2.24.49 CXCL1.25.26 CCR2.26.12 CCR6.25.13 CXCR4.5.24 C3R.49.49 C5R.5.25 REM-1.49.24 LR1.24.24 LR2.26.25 LR4.25.12 LR5.51.51 LR6.27.26 LR9.25.51 CD4.24.25 CD8.25.25 CD86.5.49 Eighteen-month-old C57BL/6 mice were microinjected with BSA (control) or Del-1 in the pltl gingiv etween the first nd the second molr teeth. he mice were then orlly inoculted with 1 9 CFU Porphyromons gingivlis in 2% croxymethylcellulose (CMC) or CMC only, nd were scrificed 12h lter. he interdentl gingiv (i.e., etween first nd second molr teeth) were dissected nd processed for qpcr to determine gingivl mrna expression of the indicted molecules (normlized ginst GAPDH mrna nd expressed s fold chnge of Del-1 treted reltive to corresponding BSA-treted controls). o ensure enough tissue mteril, dissected interdentl gingiv from 5 mice per group were pooled efore use in the qpcr. Consistent results were otined from n identicl independent experiment.
19 SUPPLEMENARY NOE Immunohistochemistry. Mxille with intct surrounding tissue were fixed in 4% prformldehyde, declcified in Immunocl solution (Decl) for 15 dys, nd emedded in OC compound. Seril mesio-distl sections (7- to 8-µm thick) prllel to the long xis of the teeth (sgittl) were stined with monoclonl ntiodies to mouse Ly6G (RB6-8C5, FICconjugte; LifeSpn BioSciences), mouse CD4 (GK1.5, FIC- or PE-conjugte; LifeSpn), mouse γδ -cell receptor (GL3, FIC-conjugte; LifeSpn Biosciences) or with polyclonl ntiodies to humn/mouse Del-1 (Proteinech), humn/mouse IL-17A (Snt Cruz Biotech), humn/mouse NF (Acm), humn/mouse RANKL (Snt Cruz), nd mouse CD31 (LifeSpn). Where necessry, stining involved the use of secondry regent (AlexFluor594-conjugted got nti-rit IgG; Moleculr Proes). he specificity of stining ws confirmed y using pproprite FIC-conjugted isotype controls or norml rit IgG followed y AlexFluor594- got nti-rit IgG. Imges were cptured using lser-scnning confocl microscope (Olympus FV1). Fluorescence intensity ws quntified using the Imge J softwre (NIH; http://rs.info.nih.gov/ij). Colocliztion nlysis. he degree of colocliztion of vrious cell types with IL-17 in confocl sections of interdentl gingiv ws quntified using the pulic domin ImgeJ softwre with the Intensity Correltion Anlysis plugin (NIH; http://rs.info.nih.gov/ij). Histologicl RAP stining. Upper jws (mxille) with intct surrounding tissue were fixed in 4% prformldehyde, declcified in Immunocl solution (Decl Chemicl Corp.) for 15 dys, nd emedded in OC compound. Osteoclsts were identified in mesio-distl sections (7- to 8-
2 µm thick) of mxille, using trtrte-resistnt cid phosphtse (RAP) stining. his ws crried out using the leukocyte cid phosphtse kit s per the mnufcturer s protocol (Sigm Aldrich). Slides were viewed using Nikon E8 microscope. RAP + multinucleted cells lying long the lveolr one surfce were considered to e osteoclsts. Histochemicl detection of β-glctosidse enzymtic ctivity. β-glctosidse ctivity in the gingiv of Edil3 -/- mice (Del-1 knock-out/lcz knock-in trnsgenics) ws detected using β - glctosidse stining kit (Mirus). Gingivl specimens, collected from mouse mxille, were frozen t -8 C in OC compound until sectioning. For stining, 7- to 8-µm thick frozen gingivl tissue sections were conditioned in PBS, fixed with 2% glutrldehyde, nd stined with X-gl solution, using the mnufcturer s protocol. Imges were cquired using Nikon E8 microscope. β-glctosidse ws lterntively detected y confocl immunohistochemistry (see Immunohistochemistry in min pper) using ma to β-glctosidse (Clone GAL-13) from Sigm-Aldrich. MPO ssy. he concentrtion of MPO in gingivl tissue homogentes ws determined using n ELISA kit ccording to the mnufcturer s instructions (Hycult Biotechnology). MPO concentrtions were normlized to the totl protein concentrtions in the tissue homogentes, s mesured using the Coomssie Plus Brdford protein ssy kit (Pierce). Quntittive rel-time PCR (qpcr). otl RNA ws extrcted from excised gingivl tissue or cultured cells using the PerfectPure RNA cell kit (5 Prime, Fisher) nd quntified y spectrometry t 26 nd 28 nm. he RNA ws reverse-trnscried using the High-Cpcity cdna Archive kit (Applied Biosystems) nd qpcr with cdna ws performed using the ABI
21 75 Fst System, ccording to the mnufcturer's protocol (Applied Biosystems). qmn proes, sense primers, nd ntisense primers for qpcr of genes investigted in this pper were purchsed from Applied Biosystems. Isoltion of mouse neutrophils. Mouse neutrophils were otined y hrvesting the one mrrow 1. Briefly, one mrrow cells, otined fter flushing tiis nd femurs with RPMI 164 contining 1% FBS, were depleted of tissue deris nd erythrocytes nd were resuspended in 45% Percoll. he cell suspension ws then overlid onto four-lyer Percoll grdient (5%, 55%, 62% nd 81%) nd centrifuged t 1,2 g for 3 min t 4 o C. Mture neutrophils were collected t the 81% interfce nd wshed twice in PBS. Neutrophil purity ws routinely > 95%, s determined y FACS fter stining for Ly6G, F4/8, nd CD3. Bone loss in ntiiotic-treted mice. In certin experiments, one loss (determined s outlined in the min pper under Determintion of periodontl one loss) ws mesured in mice which were dministered ntiiotics in their drinking wter. Specificlly, the mice were provided wter supplemented with sulfmethoxzole nd trimethoprim t finl concentrtion of 8 µg/ml nd 4 µg/ml, respectively. Control mice were given plin wter. rnsmigrtion ssy. rnsmigrtion ssys were performed s previously descried 1, using 6.5-mm rnswells with n 8-µm pore size (Corning). Briefly, humn umilicl vein endothelil cells (HUVEC; PromoCell) were cultivted on rnswell filters 2 dys prior to the ssy nd grown without medium in the lower comprtment for 48 h (37 C, 5% CO 2 ). Neutrophils isolted from humn peripherl lood were dded to the upper well nd were llowed to migrte towrds migrtion-ssy medium (serum-free RPMI with.3% BSA in the sence or presence of 2
22 ng/ml CXCL8) in the lower comprtment of the rnswell system. After 6 min incution t 37 C, the numer of trnsmigrted cells in the lower comprtment ws mesured 1. Solule Del-1 used in these experiments ws from R&D Systems. Humn lood collections were conducted in complince with estlished guidelines pproved y the Institutionl Review Bord of the University of Louisville. Chemotxis. he chemotxis of primry neutrophils isolted from the one mrrow of wild-type or Edil3 -/- mice towrds 1 ng/ml of CXCL2 ws tested using rnswell system with 5-µm pores (Corning) ccording to previously descried protocol 2. Peripherl quntittive computer tomogrphy (pqc). he left femur nd lumr spine, stored in 7% ethnol, were used for one minerl density (BMD) mesurements. BMD of the distl femorl metphysis nd diphysis ws mesured y pqc using XC Reserch M+ pqc mchine (Strtec Medizintechnik). he mesurements were mde with voxel size of 7 µm. One slice in the mid-diphysis of the femur locted 7 mm distl from the growth plte, nd three slices in the femorl metphysis locted 1 mm distl from the growth plte were mesured. he position of the growth plte ws determined with the help of scout view imge. For the mesurement of treculr BMD, regions of interest were set nd contour mode 1 nd peel mode 2 were used. he threshold for the treculr one ws 23 mg/cm³ nd the threshold for corticl BMD ws 71 mg/cm 3. Humn gingivl tissue smples: Gingivl tissue smples were otined during periodontl surgery under Institutionl Review Bord pprovl (University of Louisville). he disese sttus ws determined ccording to the Armitge periodontl disese clssifiction 3. he ptients (4
23 mles nd 3 femles, ge rnge etween 4 nd 5) presented loclized severe chronic periodontitis. Ptients with uncontrolled dietes, previous hed nd neck rdition therpy, chemotherpy in the previous 12 months immune diseses or other systemic diseses tht significntly ffect the periodontium, were excluded from the study. Smoker nd pregnnt ptients were lso excluded from the study. Disesed (inflmed) tissues were collected round involved teeth nd corresponded to ttchment loss 5 mm nd proing depth 5 mm. Control sites were free of pprent periodontl inflmmtion nd they corresponded to zero ttchment loss nd proing depth 3 mm; these were dignosed s periodontlly helthy sites nd were seprted y t lest one tooth from periodontl inflmmtory lesions. 1. Orlov, V.V. et l. A novel pthwy of HMGB1-medited inflmmtory cell recruitment tht requires Mc-1-integrin. EMBO J. 26, 1129-1139 (27). 2. Wolf, R. et l. Gene from psorisis susceptiility locus primes the skin for inflmmtion. Sci. rnsl. Med. 2, 61r9 (21). 3. Armitge, G.C. Periodontl dignoses nd clssifiction of periodontl diseses. Periodontol. 2 34, 9-21 (24).