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VMI
 


Vascular Medicine Institute
University of Pittsburgh
BST E1240
200 Lothrop Street
Pittsburgh, PA 15261
Phone: 412-383-5853
Fax: 412-648-5980

Prithu Sundd, PhD

 

prithu sundd phd

 

Prithu Sundd, PhD

Assistant Professor of Medicine,
Division of Pulmonary, Allergy and Critical Care Medicine


Assistant Professor of Bioengineering

E1255 BST
200 Lothrop Street
Pittsburgh, PA 15261

Office phone: 412-648-9103
Lab phone: 412-648-9987
Email: prs51@pitt.edu


Sundd Lab

   

Education and Training

PhD, 2008, Chemical Engineering, Ohio University, Athens, OH

Postdoctoral Research, 2013, Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA

Postdoctoral Research, 2013, Laboratory of Microfluidics, Department of Physics, University of California San Diego, San Diego, CA

Research Interests

1. Mechanisms of leukocyte rolling and arrest during inflammation.

Inflammatory response following bacterial infection involves neutrophil adhesion to the inflamed endothelium of blood vessels with high wall shear stress (t > 6 dyn/cm2). Neutrophil-endothelial adhesion starts with rolling along the vessel wall mediated by P-selectin on the endothelium binding to P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils, followed by firm arrest which is mediated by activated β2-integrins (LFA-1 and Mac-1) on the neutrophil binding to inter-cellular-adhesion-molecule-1 (ICAM-1) on endothelium. In October 2010 (Sundd, P. et al. Nature Methods, 2010), I introduced quantitative Dynamic Footprinting (qDF) microscopy which is an adaptation of TIRF microscopy and allows estimation of z-distances in the footprints (cell-substrate contact zone) of rolling neutrophils. This study revealed that neutrophils rolling at high shear stress (> 6 dyn/cm2) deform creating a four-fold larger footprint with the P-selectin substrate than that predicted by computational models and low resolution in vivo images, and that the rolling is further facilitated by three to four long membrane tethers which can extend up to 16 µm behind the rolling cell. In the most recent study (Sundd P. et al, Nature, 2012), I have discovered ‘sling’, an autonomous adhesive structure made by rolling neutrophils. I have shown that long tethers made by neutrophils rolling at high shear stress (6-10 dyn/cm2) do not retract as postulated, but instead persist and appear as ‘slings’ at the front of rolling neutrophils (Movie 1). Slings are made by rolling neutrophils in vitro and in a model of acute inflammation in vivo. Selectin ligand PSGL-1 is presented as discrete sticky patches while integrin LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling neutrophils and undergo a step-wise peeling from the P-selectin substrate which is enabled by the tandem failure of PSGL-1 patches under hydrodynamic forces (Movie 2). Currently, we are conducting experiments in an in vitro microfluidic flow chamber to elucidate the cytoskeletal organization responsible for the ability of slings to withstand hydrodynamic forces at high shear stresses.
We are also conducting qDF experiments to study the nature of sling formation by different mouse circulating monocyte subsets (Gr-1+/Ly6Chi and Gr-1-/Ly6Clow) and their role in monocyte adhesion during inflammation.

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During bacterial infection neutrophils leave the blood stream and enter the infected tissue to resolve infection. In order to leave the blood stream, neutrophils have to first roll along the walls of blood vessels. In some blood vessels, the blood flow is very fast, however, neutrophils still manage to roll along the vessel wall and enter the infected tissue. We discovered that neutrophils form long tube like structures known as ‘slings’ that help them to roll in presence of fast blood flow. Top panel-animation showing the side view of a rolling neutrophil. The rolling neutrophil forms a ‘sling’ in the front and then wraps it around. Bottom panel-experimental movie showing a mouse neutrophil forming a sling in the front. The cell is rolling on a P-selectin coated cover glass in a microfluidic device and the movie was recorded using qDF. The view is from the bottom of the cell. The animation in the top panel is inspired by the experimental movie shown in the bottom panel. Sundd P. et al. Nature 488:399-403,2012.

 

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Step-wise peeling of a sling. PSGL-1 patches (red spots) visible on sling (green). TIRF excitation 561 and 488 nm, incidence angle θ = 70⁰. P-selectin 20 molecules/µm2. Wall shear stress 10 dyn/cm2. View from the bottom. Frame rate 5 s-1. Isolated mouse bone marrow neutrophils were stained with membrane dye DiO and AlexaFluor568-conjugated nonblocking Ab against PSGL-1. Cells were allowed to roll on P-selectin coated cover glass in a microfluidic device and images were recorded using dual color qDF (DqDF).

 

2. Role of neutrophils in pulmonary vaso-occlusion during sickle cell disease Acute Chest Syndrome.

Sickle cell disease (SCD) is an autosomal recessive point mutation in the beta globin gene, generating a mutant hemoglobin S (Hb-S) molecule that will polymerize when deoxygenated. Accumulation of intra-erythrocytic Hb-S polymer leads to cellular rigidity, altered rheological properties, expression of surface integrins and other adhesion molecules (ICAM-4, VLA-4, CD36, sulfated glycolipids and tetrasaccharides), and ultimate entrapment of the cells in the microcirculation. As a result, the microcirculation can become occluded in SCD patients, producing ischemia and reperfusion injury to almost any organ in the body. This occurs frequently in the lung, where red cell-leukocyte-endothelial adhesive interactions can cause a form of acute lung injury called the Acute Chest Syndrome (ACS). While the mechanisms of vaso-occlusion have been characterized in the systemic circulation, little is known about the molecular vaso-occlusive events in the lung microcirculation. Unlike systemic vasculature where leukocyte recruitment takes place primarily in the postcapillary venules, pulmonary capillaries are the primary site of leukocyte sequestration in lungs during bacterial infection. However, several factors are likely to contribute to pulmonary vaso-occlusion including the pro-adhesive properties and enhanced rigidity of sickle RBCs, the large marginated pool of neutrophils in the pulmonary capillaries, and the hyper-inflammatory state present in SCD. We are using multi-photon intra-vital microscopy to study how inflammation drives pulmonary vaso-occlusion in SCD mice.

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Visualization of the pulmonary microcirculation and alveoli in a live C57BL/6 mouse. The pulmonary capillaries surrounding the alveoli can be seen moving in and out of the imaging plane in the z-direction due to the dynamic expansion and contraction of the alveoli with mechanical ventilation. Intravascular FITC dextran highlights the pulmonary capillaries and a feeding arteriole in green. The feeding arteriole has a diameter of 33 µm, while the alveolar capillaries have an average diameter of 6 ± 2 µm. 10x of original acquisition rate. Bennewitz, M. F., Watkins, S. C. & Sundd, P. IntraVital 3, e29748, (2014).

 

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Elongated neutrophils slowly transit through the pulmonary capillaries of a live C57BL/6 mouse. Neutrophils (green) were stained by intravascular (iv) injection of FITC Ly-6G mAb.. Texas Red-conjugated dextran (red) was administered iv to visualize pulmonary capillaries and a feeding arteriole. Neutrophils take on an elongated shape that fills the lumen of pulmonary capillaries.  The feeding arteriole has a diameter of 21 µm, while the capillaries have an average diameter of 6 ± 2 µm. Bennewitz, M. F., Watkins, S. C. & Sundd, P. IntraVital 3, e29748, (2014).


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RBC and neutrophil trafficking in the pulmonary microcirculation of live BERK SCD mice in the absence of an inflammatory stimulus. RBCs are shown in green, neutrophils in red, and the pulmonary microcirculation in purple. The feeding arteriole has a diameter of 41 µm, while the capillaries have an average diameter of 7 ± 2 µm. 2.5x of original acquisition rate. Bennewitz, M. F., Watkins, S. C. & Sundd, P. IntraVital 3, e29748, (2014).

 

3. Identifying molecular mechanism of vaso-occlusion in SCD (SS) patient blood.

Sickle Cell Disease (SCD) is an autosomal-recessive-hemolytic disorder caused by a single point mutation in the β-globin gene that leads to sickling of RBCs under deoxygenated condition. Sickle RBCs (sRBCs) are not only rigid but also express adhesion molecules, which are not normally expressed on RBCs. The sticky and rigid sRBCs are believed to get trapped in blood vessels along with leukocytes to cause vaso-occlusion, which is the predominant pathophysiology underlying acute pain crisis and emergency medical care among SCD patients. The process of sickling and vaso-occlusion also leads to sRBC hemolysis, which releases hemoglobin, ADP and other RBC contents into the blood giving rise to a pro-inflammatory and pro-coagulant state. Although neutrophils have been shown to play a role in the onset of vaso-occlusion by interacting with sRBCs and platelets in cremaster venules of SCD mice; the cellular, molecular and biophysical mechanisms that enable vaso-occlusion in SCD (SS) patients are not fully understood. We are currently conducting experiments with SS patient blood to understand the molecular and biophysical mechanism of interaction between individual blood cells that leads to vaso-occlusion. Freshly collected heparinized blood from SCD (SS) patients or race matched control subjects is perfused through PDMS based microfluidic micro-channels with a glass bottom either cultured with human micro-vascular endothelial cells or coated with a cocktail of recombinant human P-selection, ICAM-1 and IL-8 at a venular/arteriolar wall shear stress. Fluorescent Abs are added to the blood to stain neutrophils, sRBCs and platelets, respectively, and cellular interactions are recorded using multi-color Quantitative Dynamic Footprinting or epifluorescence microscopy.

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Transition from rolling to arrest on P-selectin, ICAM-1, and IL-8 coated substrate in SS patient blood. Wall shear stress 6 dyn cm-2. Neutrophils (violet; AF647-anti-CD16 Ab). Excitation laser- 640 nm. Imaging step-1. 50x of original frame rate. Jimenez et al, Haematologica. 2015 May 14. pii: haematol.2015.126631.

 

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Neutrophils rolling and arresting on TNF-α activated human lung microvascular endothelial cells (HMVECs-L) in control human blood. HMVECs-L (green; PE-anti-PECAM-1 Ab). Neutrophils (violet; AF647-anti-CD16 Ab). Wall shear stress 6 dyn cm-2. Excitation laser- 640 and 561 nm. 10x of original frame rate. Jimenez et al, Haematologica. 2015 May 14. pii: haematol.2015.126631.


Publications

 
 
 
 
 
 
 
 
 
   

Jimenez MA, Novelli EM, Shaw GD, Sundd P. Glycoprotein Ibα inhibitor (CCP-224) prevents neutrophil-platelet aggregation in sickle cell disease. Blood Advances, 2017.

Bennewitz MF, Jimenez MA, Vats R, Tutuncuoglu E, Jonassaint J, Kato GJ, Gladwin MT, Sundd P. Lung vaso-occlusion in sickle cell disease mediated by arteriolar neutrophil-platelet microemboli. JCI Insight, 2017.

Jimenez MA, Tutuncuoglu E, Barge S, Novelli EM, Sundd P. Quantitative microfluidic fluorescence microscopy to study vaso-occlusion in Sickle Cell Disease. Haematologica, 2015.

Bennewitz MF, Watkins SC, Sundd P. Quantitative intravital two-photon excitation microscopy reveals absence of pulmonary vaso-occlusion in unchallenged Sickle Cell Disease mice. IntraVital 3, e29748, 2014.

Koltsova EK, Sundd P, Zarpellon A, Ouyang H, Mikulski Z, et al. Genetic deletion of platelet glycoprotein Ib alpha but not its extracellular domain protects from atherosclerosis. Thromb Haemost 112, 2014.

Stadtmann A, Germena G, Block H, Boras M, Rossaint J, Sundd P, Lefort C, Fisher CI, Buscher K, Gelschefarth B, Urzainqui A, Gerke V, Ley K, Zarbock A. The PSGL-1-L-selectin signaling complex regulates neutrophil adhesion under flow. J Exp Med, 2013.

Sundd P, Ley K. Quantitative dynamic footprinting microscopy. Immunology and Cell Biology, 2013 Apr;91(4):311-20.

Sundd P, Pospieszalska MK, Ley K. Neutrophil rolling at high shear: flattening, catch bond behavior, tethers and slings. Molecular Immunology, 2013 Aug;55(1):59-69.

Sundd P, Gutierrez E, Koltsova EK, Kuwano Y, Fukuda S, Pospieszalska MK, Groisman A, Ley K. ‘Slings’ enable neutrophil rolling at high shear. Nature 488:399-403,2012.

Sundd P, Pospieszalska MK, Cheung LS, Konstantopoulos K, Ley K. Biomechanics of leukocyte rolling. Biorheology48:1-35, 2011. A figure from this manuscript was on the cover page of Biorheology.

Sundd P, Gutierrez E, Petrich B, Ginsberg MH, Groisman A, Ley K. Live cell imaging of paxillin in rolling neutrophils by dual-color quantitative dynamic footprinting (DqDF). Microcirculation18(5): 361-372, 2011. Images from this study were on the cover page of Microcirculation.

Gutierrez E, Tkachenko E, Besser A, Sundd P, Ley K, Danuser G, Ginsberg MH and Groisman A. High Refractive Index Silicone Gels for Simultaneous Total Internal Reflection Fluorescence and Traction Force Microscopy of Adherent Cells. PLoS One6(9), 2011, e23807.       

Sundd P, Gutierrez E, Pospieszalska MK, Zhang H, Groisman A, Ley K. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nature Methods 7:821-824, 2010. Images from this study were on the cover page of the October, 2010 issue of Nature Methods.

Sundd P, Zou X, Goetz DJ, and Tees DFJ. Leukocyte adhesion in capillary-sized, P-selectin-coated micropipettes. Microcirculation 15: 109-122, 2008.

Pai A, Sundd P, Tees DFJ. In situ microrheological determination of neutrophil stiffening following adhesion in a model capillary. Ann Biomed Eng 36: 596-603, 2008.

Book Chapters

Sundd, P. & Bennewitz, M.F. Leukocyte kinetics and migration in the lungs. in Hematologic Abnormalities and Acute Lung Syndromes (eds. Lee, J.S. & Donahoe, M.P.) 19-45 (Springer International Publishing, Switzerland, 2017). 10.1007/978-3-319-41912-1.

Ley K, Mestas J, Pospieszalska MK, Sundd P, Groisman A, and Zarbock A. Intravital microscopic investigation of leukocyte interactions with the blood vessel wall. In: Methods in Enzymology: Elsevier Academic Press, 445, 2008, p. 255-79.

Tees DFJ, Sundd P, Goetz DJ. A flow chamber for capillary networks: Leukocyte adhesion in capillary-sized, ligand-coated micropipettes. In: Principles of cellular engineering: Understanding the biomolecular interface, edited by M. R. King. New York: Academic Press, 2006.

Pubmed link