1. Acute lung injury in Sickle Cell Disease
Acute lung injury in Sickle Cell Disease. Sickle Cell Disease (SCD) affects ~100,000 Americans and millions world-wide. Vaso-occlusion or blockage of blood vessels by blood cell aggregates is the predominant pathophysiology in SCD. Acute systemic painful vaso-occlusive episode, which is the primary reason for emergency medical care among SCD patients, is often an antecedent to acute chest syndrome (ACS), a type of acute lung injury. ACS is among the leading causes of mortality in SCD but the current treatment for ACS is primarily supportive and the etiological mechanism remains largely unknown. My lab is using a multiscale-integrative-physiologic approach involving multi-photon-excitation intravital microscopy of intact lung in live transgenic humanized SCD mice and live-cell fluorescence microscopy of SCD patient blood flowing through a microfluidic platform in vitro. We have found (Bennewitz et al, JCI-Insight 2017) that vaso-occlusive episode triggered entrapment of P-selectin dependent platelet-neutrophil embolic aggregates in pulmonary arterioles, leading to arrest of blood flow in the lung of SCD mice. Our recent work (Vats et al, AJRCCM 2019) identifies a role for platelet-inflammasome and IL-1β carrying platelet extracellular vesicles in promoting lung vaso-occlusion in SCD. Our findings suggest that inhibitors of inflammasome or IL-1β dependent innate immune pathway can be beneficial in preventing ACS of SCD.
Movie #1: https://insight.jci.org/articles/view/89761/sd/8
Legend: Pulmonary vaso-occlusions (white circles) blocking all 4 arteriolar bottle-necks in a SCD mouse administered 0.1 μg/kg IV LPS. Neutrophil vaso-occlusions (red arrows). Platelet vaso-occlusions (green arrows). White arrow-direction of blood flow. Pulmonary microcirculation (purple). 2/3x original acquisition rate. Movie captured using quantitative Fluorescence Intravital Lung Microscopy (qFILM). Bennewitz and Jimenez et al, Journal of Clinical Investigation Insight. 2017;2(1):e89761.
Movie #2: https://insight.jci.org/articles/view/89761/sd/7
Legend: Neutrophils (red) bound to platelets (blue) are occluding the arteriolar bottleneck in a SCD mouse administered 0.1 μg/kg IV LPS. Erythrocytes (green) are stationary downstream of the vaso-occlusion but erythrocytes upstream of the vaso-occlusion are colliding with the aggregate and then bypassing through the side branch of the arteriole. White arrow-direction of blood flow. Pulmonary microcirculation (purple). 1/3x original acquisition rate. Movie captured using quantitative Fluorescence Intravital Lung Microscopy (qFILM). Bennewitz and Jimenez et al, Journal of Clinical Investigation Insight. 2017;2(1):e89761.
Movie #3: https://insight.jci.org/articles/view/89761/sd/18
Legend: Freely flowing platelets interacting with arrested neutrophils in control human blood perfused through microfluidic micro-channels presenting P-selectin, ICAM-1 and IL-8. Movie captured using quantitative Microfluidic Fluorescence Microscopy (qMFM). Aqcquisition (10 frames s-1). Neutrophils (purple). Platelets (green). Wall shear stress 6 dyn cm-2. Bennewitz and Jimenez et al, Journal of Clinical Investigation Insight. 2017;2(1):e89761.
Movie #4: https://insight.jci.org/articles/view/89761/sd/19
Legend: Freely flowing platelets interacting with arrested neutrophils in SCD human blood perfused through microfluidic micro-channels presenting P-selectin, ICAM-1 and IL-8. Movie captured using quantitative Microfluidic Fluorescence Microscopy (qMFM). Acquisition (10 frames s-1). Neutrophils (purple). Platelets (green). Wall shear stress 6 dyn cm-2. Bennewitz and Jimenez et al, Journal of Clinical Investigation Insight. 2017;2(1):e89761.
Movie #5: https://insight.jci.org/articles/view/89761/sd/20
Legend: 360-degree view of the super resolution Structured Illumination Microscopy (SIM) image showing the distribution of F-actin (purple) and P-selectin (blue) on platelets attached to an arrested neutrophil. SCD human (steady state) blood perfused through microfluidic micro-channels presenting P-selectin, ICAM-1 and IL-8 and fixed under flow. Wall shear stress 6 dyn cm-2. Bennewitz and Jimenez et al, Journal of Clinical Investigation Insight. 2017;2(1):e89761.
Legend: Platelet NLRP3-inflammasome and IL-1β innate immune pathway promotes lung vaso-occlusion in SCD. The inflammatory milieu in SCD (DAMPs) primes TLR4-dependent activation of NLRP3-ASC-Caspase-1 inflammasome in platelets (green), which is enhanced by the presence of TLR4 agonists (PAMPs) at low concentrations that are innocuous under healthy conditions. Inflammasome dependent Caspase-1 activation promotes platelet activation (black curved arrow), leading to shedding of IL-1β and Caspase-1 carrying extracellular vesicles (EVs; shown in yellow) by platelets. Platelet EVs promote IL-1β and Caspase-1 dependent platelet-neutrophil aggregation in lung arterioles (black solid arrows) leading to pulmonary vaso-occlusion. Previously, we identified that platelet-neutrophil aggregation dependent pulmonary vaso-occlusion can be prevented by a P-selectin blocker (gray block line). Here, we show that inhibiting NLRP3-inflammasome or IL-1β innate immune pathway (black block lines) prevents lung vaso-occlusion in SCD. Although not shown in our current study, IL-1β carrying platelet EVs may activate the IL-1 receptor on platelets by an autocrine loop to further promote generation of platelet EVs (gray curved arrow). Also, activated platelets trapped within the platelet-neutrophil aggregates may undergo degranulation to locally generate IL-1β and Caspase-1 carrying EVs (gray dotted arrow). SCD, Sickle Cell Disease; DAMPs, Damage Associated Molecular Patterns; PAMPs, Pathogen Associated Molecular Patterns; NLRP3, Nod-Like Receptor family, Pyrin domain containing 3; ASC, Apoptosis-associated Speck-like protein containing a Caspase recruitment domain; IL-1β, Interleukin-1β; Cas-1, Caspase-1; EV, Extracellular Vesicle; TLR4, Toll-Like-Receptor- 4. Vats, Brzoska, Bennewitz and Jimenez et al, Am J Respir Crit Care Med. 2020 Jan 1;201(1):33-46.
2. Cigarette smoke induced flu severity
Cigarette smoke induced flu severity. Cigarette smoking has been associated with development of flu induced acute lung injury (ALI). However, the innate immune pathways that leaves cigarette smokers at risk of developing flu-triggered ALI remains poorly understood. We have developed two-hit model in mice that involves cigarette smoke exposure followed by intranasal instillation of influenza flu virus. We are using intravital microscopy of lung in mice, biochemical approaches and in vitro studies with patient blood samples to identify the role of neutrophil-platelet aggregates and how innate immune signaling in these cells contribute to the pathogenesis of cigarette smoke induced severity of flu infection.
3. Joint-injury in hemophilia
Joint-injury in hemophilia. Hemarthrosis is a major complication of hemophilia and ultimately leads to debilitating-painful-arthropathy, primarily affecting elbow, knee and ankle joints. Despite the development and implementation of factor replacement therapies that prevent acute joint bleeding, these events continue to occur, and the current therapy is limited to target joint-replacement surgery. My lab is conducting in vitro studies with hemophilia-A patient blood samples and in vivo studies in FVIII deficient (hemophilia-A) mice to understand how innate immune signaling in neutrophils and platelets contribute to progression of hemophilic arthropathy.
4. Pulmonary Hypertension
Pulmonary Hypertension. Pulmonary hypertension (PH) is a debilitating condition manifested by pulmonary vascular remodeling, elevated pulmonary artery pressure, and right heart failure. One of the new areas of research in my lab is to identify whether neutrophil-platelet aggregation and platelet extracellular vesicles dependent pulmonary arteriole thrombosis contributes to progression of PH. We are conducting in vivo studies in rodents and in vitro studies with PH patient blood samples to identify the molecular pathways that promote neutrophil-platelet aggregation and thrombosis in pulmonary arterioles and test whether inhibiting these pathways at an early stage can prevent the vascular remodeling and cardiovascular morbidity manifested at later stages.
5. 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.
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.
6. Biology of hemolysis-triggered pulmonary thrombosis
Biology of hemolysis-triggered pulmonary thrombosis
Patients with hereditary or acquired hemolytic anemias have a high risk of developing in-situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Recently (Brzoska et al JCI-Insight 2020), we used intravital lung microscopy in mice for the first time to assess the pathogenesis of pulmonary thrombosis following deionized-water induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow (movie #1 below). The hemolysis-induced pulmonary thrombosis was phenocopied with intravenous ADP- (movie #2 below) but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet-P2Y12 purinergic-receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation leading to thrombosis in the pre-capillary pulmonary arterioles and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.
Movie #1: https://insight.jci.org/articles/view/139437/sd/3
Legend: Transient pulmonary thrombosis in WT mouse following 150 µl IV dH2O. Platelets (green) and pulmonary microcirculation (purple). t = 0 s corresponds to time before and t > 0 s correspond to time following IV dH2O administration, respectively. White arrow-direction of blood flow within the feeding arteriole.
Movie #2: https://insight.jci.org/articles/view/139437/sd/10.
Legend: Transient pulmonary thrombosis in WT mouse following 2.5 mg/kg IV ADP. Platelets (green) and pulmonary microcirculation (purple). t = 0 s corresponds to time before and t > 0 s correspond to time following IV ADP administration, respectively. White arrow-direction of blood flow within the feeding arteriole. 1.5x original acquisition rate.