Research Interests
1. 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.
