Pulmonary Thrombo-inflammation in Sickle Cell Disease
Sickle cell disease (SCD) affects more than 100,000 Americans and millions more worldwide. Vaso-occlusion, or blockage of blood vessels by blood cell aggregates, is the predominant pathophysiology in SCD. Acute systemic, painful vaso-occlusive episodes, which are the primary reasons 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.
The Sundd Lab uses a multiscale-integrative-physiologic approach involving multi-photon-excitation intravital (in vivo) microscopy of intact lung in live, transgenic, humanized SCD mice and SCD patient blood flowing through a microfluidic platform in vitro.
We have found (Bennewitz et al, JCI-Insight 2017, Bennewitz et al Blood Advances 2020, Vats et al Experimental Hematology 2020) that vaso-occlusive crisis triggered entrapment of P-selectin dependent platelet-neutrophil embolic aggregates in pulmonary arterioles, leading to the arrest of blood flow in the lung of SCD mice. Our recent work (Vats et al, AJRCCM 2020) 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 SCD. Our more recent findings (Vats and Kaminski et al, Blood 2022) show how sterile inflammation in SCD promotes Gasdermin-D-dependent shedding of neutrophil extracellular traps (NETs) in the liver and how these NETs travel intravascularly (embolize) from the liver to the lung, to promote P-selectin-independent lung vascular vaso-occlusion in SCD.
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.
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.
Lung intravital microscopy reveals neutrophil extracellular traps (NETs) in the pulmonary arteriole bottle-neck of an SCD mouse IV administered 10 µmole/kg oxy-Hb. Pulmonary microcirculation (purple), neutrophils (blue), extracellular DNA (green), and citrullinated histones (red). Arrow denotes direction of blood flow. Scale bar 20 µm. 1/3x original acquisition rate. Movie captured using quantitative Fluorescence Intravital Lung Microscopy (qFILM). Vats R and Kaminski TW et al, Blood. 2022 Sep 1;140(9):1020-1037. doi: 10.1182/blood.2021014552.
Lung intravital microscopy reveals circulating NETs (green) entering the lung microcirculation (purple) via the pulmonary arteriole in an SCD mouse IV administered 10 µmole/kg oxy-Hb. Neutrophils (red) and extracellular DNA (green). Arrow denotes direction of blood flow. Scale bar 20 µm. 1/3x original acquisition rate. Movie captured using quantitative Fluorescence Intravital Lung Microscopy (qFILM). Vats R and Kaminski TW et al, Blood. 2022 Sep 1;140(9):1020-1037. doi: 10.1182/blood.2021014552.
Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ pulmonary arteriole thrombosis (iPAT). While pulmonary thrombosis is a major morbidity risk 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 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.
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 were the first in vivo studies to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles. Currently, we are conducting further studies to understand the role of platelet purinergic signaling in the pathogenesis of hemolysis-induced pulmonary thrombosis.
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 denotes direction of blood flow within the feeding arteriole. 1.5x original acquisition rate. Movie captured using quantitative Fluorescence Intravital Lung Microscopy (qFILM). Brzoska et al, JCI Insight. 2020 Jul 23;5(14):e139437. doi: 10.1172/jci.insight.139437.
Cigarette smoking has been associated with the development of flu-induced acute lung injury (ALI). However, the innate immune pathways that leave cigarette smokers at risk of developing flu-triggered ALI remain poorly understood. We have developed a 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 contributes to the pathogenesis of cigarette smoke-induced severity of flu infection.
Hemarthrosis (joint bleeding) is a major complication of hemophilia and ultimately leads to debilitating, painful hemophilic 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. The Sundd Lab is conducting in vitro studies with hemophilia A patient blood samples and in vivo studies in FVIII deficient (hemophilia A) mice to understand the role of inflammasome-dependent, innate immune signaling in promoting hemophilic arthropathy. We hypothesize that bleeding of a synovial joint results in local hemolysis with heme-driven inflammation and ultimate joint destruction, and that therapies targeting inflammasome pathway will alleviate the joint inflammation and prevent long-term joint damage. Currently, experiments are underway to identify the role of eDAMPs (heme and hemoglobin) mediated activation of TLR4 and inflammasome pathway in promoting IL-1b generation, neutrophil-platelet aggregation, neutrophil extracellular traps (NETs) release and progression of joint injury in hemophilia A mice. Based on the preliminary findings, Dr. Sundd recently received the prestigious 2021 Bayer Hemophilia Research Award from the Bayer Hemophilia Award Program.