Hemolysis and Immune Activation. Karina Yazdanbakhsh, PhD Executive Director, Research Institute New York Blood Center

Transcription

1 Hemolysis and Immune Activation Karina Yazdanbakhsh, PhD Executive Director, Research Institute New York Blood Center

2 Hemolysis in Sickle Cell Disease (SCD) Hemolysis activates the underlying endothelium: increased expression of endothelial adhesion molecules and apoptotic markers Attachment of sickle RBCs and other blood components to the vessel wall; in vivo heme injection induces vascular stasis and acute chest syndrome Dutra, F. F. and M. T. Bozza (214). Front Pharmacol 5: 115. Anti-inflammatory; Anti-cytotoxic Heme oxygenase 1 (HO-1) breaks down heme upregulated in SCD Balla et al Proc Natl Acad Sci U S A.;9(2): Belcher et al. 214 Blood;123(3): Camus, et al. 215 Blood;125(24): Belcher et al.26 J Clin Invest; 116(3):88-16 Gosh et al 213 J Clin Invest: 123(11): Hoover et al. Blood. 1979;54(4): Hebbel et al 198;32(18): Hebbel et al J Clin Invest. 198;65(1): Hebbel et al Blood. 1981;58(5): Mohandas N, Evans E. Blood. 1984;64(1): Heme scavenging/removal system (hemopexin and haptoglobin) is overwhelmed

3 Patrolling Monocytes Phagocytose cellular debris derived from damaged endothelial cells Control endothelial damage in atherosclerosis models and clear vascular amyloid beta in Alzheimer s disease SCD express high levels of HO-1 in patrolling monocytes: control T cell anti-inflammatory profile in SCD under hemolytic conditions (Zhong Yazdanbakhsh, (214) JI 193(1):12-1) Carlin et al. (213) Cell 153(2): Quintar et al. (217) Circ Res 12(11): Michaud et al. (213) Cell Rep 5(3): Zhong Yazdanbakhsh. (214). J Immunol 193(1): Hypothesis: HO-1 expressing patrolling monocytes clear heme damaged endothelial cells and sickle RBC attached to ECs in SCD, dampening inflammation

4 M F I o f m a r k e r (R a la tiv e to H D ) HO-1 expressing Patrolling Monocyte Characterization in SCD M F I o f H O HO-1 MFI H D S C D H D S C D H D S C D H D S C D T o t a l m o n o C la s s ic a l In t e r m e d ia t e P a tr o llin g % H O -1 h i m o n o c y t e HO-1 hi H D S C D H D S C D H D S C D H D S C D T o ta l C la s s ic a l In t e r m e d i a t e P a t r o llin g H O -1 low H O -1 h i M F I o f T N F Expanded subpopulation of circulating patrolling monocytes expressing high levels of HO-1 in SCD C D 3 1 T ie 2 N U R 7 7 H L A -D R C D 8 6 C D 8 C D 1 1 b C D 1 4 C X 3 C R 1 H O -1 lo w H O -1 h i Liu. Yazdanbakhsh. Blood 218, 131(14):16

5 % H O -1 h i P a tr o llin g m o n o c y t e % H O -1 h i 6 4 P a tr o llin g m o n o c y t e % H O -1 h i m o n o Mechanism of HO-1 hi Upregulation in Patrolling Monocytes Liu. Yazdanbakhsh. Blood 218, 131(14):16 HD SCD Patrolling Mo H e m in / M H e m in tr e a te d H U V E C H e m in / M H e m in tr e a te d H U V E C % H O -1 h i C la s s ic a l m o n o c y t e H e m in / M H e m in tr e a te d H U V E C H e m in / M Classical Mo H e m in tr e a te d H U V E C Interaction of cell free heme with endothelial cells is prerequisite for optimal induction of HO-1 hi expression in patrolling monocytes % H O -1 h i P a tr o llin g m o n o RBC lysate % H O -1 h i P a tr o llin g m o n o c y t e M e d i u m R B C ly s a te - H U V E C M e d iu m H e m in H e m in + H P X + H U V E C + Hemopexin

6 HO-1 hi Patrolling Monocytes in SCD patients with Vaso-occlusive Crisis (VOC) s V C A M - 1 (n g /m l) N e u t r o p h il P a tr o llin g m o n o c y t e ( / l b lo o d ) H O -1 h i P a tr o llin g m o n o c y t e (/ l b lo o d ) svcam-1 Neutrophils Total monocytes 5 H D N o n V O C V O C (1 3 / l b lo o d ) H D N o n V O C V O C M o n o c y t e (1 3 / l b lo o d ) H D N o n V O C HO-1 hi patrolling Mo correlate negatively with VOC in SCD V O C Patrolling monocyte numbers HO-1 hi Patrolling monocyte numbers Liu. Yazdanbakhsh. Blood 218, 131(14):16 H D N o n V O C V O C N o n V O C V O C

7 SCD GPA HD Phagocytosed RBCs in Circulating SCD PMos CMo PMo % G P A + m o n o c y t e H D S t e a d y s t a t e S C D A c u t e c r is is S C D H D S t e a d y s t a t e S C D A c u t e c r is is S C D % P M o in m o n o c y t e H D S t e a d y s t a t e S C D A c u t e c r is is S C D CD45 BF CD45 GPA Merge P M o 91 % C M o RBC engulfed material is present in the circulating PMos of patients with SCD which is further increased during crisis, and may lead to reduced PMo numbers.

8 % C F SE + c e lls % C F SE + c e lls Sickle RBC CFSE Normal RBC Mechanism of Sickle RBCs Uptake by PMo HMVEC Medium + Hemin or PBS Culture 2 h PMo HMVEC Monocyte and CFSE labeled sickle or control RBC 2x washes Cultured overnight + Hemin Hemin-treated HMVEC Analysis using FCM and IFC CMo Medium HMVEC Hemin-treated HMVEC PMo uptake sickle RBC only when attached to ECs mostly through CD11a, CD18 and ICAM1; upregulate HO-1: cryoprotective; heme damaged ECs increase sickle RBC uptake by PMo N o r m a l R B C S ic k le R B C N o rm a l R B C + H M V E C S ic k le R B C + H M V E C N o r m a l R B C + H e m in - tr e a te d H M V E C S ic k le R B C + H e m in -tr e a te d H M V E C HO P M o HD C M o P M o SCD C M o

9 PMo Uptake Sickle RBCs In Vivo % D ii+ c e lls DiI Control RBC Sickle RBC PMo CMo WT DiI-labeled Sickle RBC Or Control RBC Hemin Flow cytometry CD11a -/- Day -1 Day +4 hours BF GFP DiI CD45 Merge CD W T m ic e + C o n tr o l R B C s W T m ic e + S ic k le R B C s C D 1 1 a -/- m ic e + S ic k le R B C s In vivo, PMo uptake sickle RBCs, but not control RBCs in part through monocyte CD11a 5 C M o P M o N e u t r ip h il L y m p h c y t e

10 PMo Uptake EC-attached Sickle RBCs In Vivo Nr4a1-GFP DiI-labeled Sickle RBC Hemin Immunofluorescence Day -1 Day +4 hours Scale = 1 µm Blue: CD31/CD144, Red: DiI, Green: GFP

11 In Vivo Effects of Sickle RBCs and Hemin in Nr4a1-/- mice WT P K H -26 la b e le d s ic k le R B C Im m u n o f lu o r e s c e n c e a n a ly s is H & E s ta in in g H e m in a n a ly s is Nra4 -/- - 1 D a y S S R B C c o u n t (.1 8 m m 2 a r e a ) W T N r 4 a 1 - /- PBS H em in Lack of patrolling monocytes drives heme-mediated endothelial activation and SCD RBC stasis Liu. Yazdanbakhsh. Blood 218, 131(14):16

12 In Vivo Effects of Sickle RBCs and Hemin in Nr4a1-/- mice GFP-Nr4a1 Nr4a -/- P K H -26 la b e le d s ic k le R B C P B S o r m o n o c y t e a d o p t iv e t r a n s f e r Im m u n o f lu o r e s c e n c e H e m in a n a ly s is - 1 d a y - 4 h S S R B C c o u n t (.1 8 m m 2 a r e a ) 1 5 P B S P a tr o llin g C la s s ic a l Patrolling monocyte protect against heme-driven endothelial activation and can inhibit hemolysis-driven SCD RBC stasis Liu. Yazdanbakhsh. Blood 218, 131(14):16

13 With Patrolling monocyte Without Patrolling monocyte Endothelial cells Hemedamaged Endothelial cells Erythrocyte Hb Heme HO-1 Patrolling monocyte Classical Mo Vasoocclusion HO-1 Endothelial cells Hemedamaged Endothelial cells

14 Protection from plasma cell-free hemoglobin and heme in sickle cell disease Victor R. Gordeuk Blood 218;131:

15 Yunfeng Liu Hui Zhong Weili Bao Woelsung Yi Vijendra Ramlall Patricia Shi Xiuli An Avital Mendelson Francesca Vinchi Montefiore Hospital Deepa Manwani Caterina Minniti Joan Uehlinger Ron Walsh Children s Hospital of Philadelphia David Friedman Stella Chou Funding Support NIH/NHLBI: R1HL R1HL13139 American Heart Association

16 In Vivo Vascular Effects of Hemin in Nr4a1 -/- Mice PBS or Hemin WT Nr4a-/- (no patrolling monocytes) 24hr Immunofluorescence (vascular activation: ICAM-1 expression on CD31/CD144+ endothelium ) WT +PBS Nra4 -/- +PBS WT +hemin IC A M - 1 A r e a / P ix e ls 2 ICAM P BS H e m in W T N r 4 a 1 - /- H O -1 + c e lls % HO-1 C la s s ic a l P BS H e m in P a t r o llin g m o n o c y t e m o n o c y t e Nra4 -/- +hemin

17 Manipulation of PMo Numbers Affects Sickle RBC Stasis In Vivo S S R B C c o u n t (.1 8 m m 2 a r e a ) MDP Clo-Lipo Control Max # P M o / l PBS Clodronate (Clo-Lipo) MDP MDP MDP Sickle mouse Day PMo Control Control CMo PMo MDP Clo-Lipo Clo-Lipo CMo Analysis PMo MDP MDP CMo Ly-6C D a y s p o s t t r e a t m e n t C o n t r o l M D P C lo - L ip o C o n tr o l M D P # C M o / l C lo -L ip o C o n t r o l M D P C lo - L ip o D a y s p o s t t r e a t m e n t # N e u t r o p h il / l C o n t r o l M D P C lo - L ip o D a y s p o s t t r e a t m e n t Control Clo-Lipo Scale = 5 µm Red: Ter-119, Green: CD31/CD144 Depletion of PMo numbers increases sickle RBC attachment to vascular endothelium and RBC stasis in SCD mice, while increasing their nos protects against tissue/organ damage Scale = 1 µm Red: F4/8, Green: CD31/CD144 1 µm Lessard, et al. Cell Rep. 217;2:183. Biburger et al. Immunity. 211;35:932