Flu Induced Acute Chest Syndrome in Sickle Cell Disease Mice

Platelet RIG-I Promotes Flu-Induced Severe Lung Injury in Sickle Cell Disease

Rationale: Sickle cell disease (SCD) affects over ∼8 million people worldwide. Acute chest syndrome (ACS), a type of acute lung injury (ALI), is a leading cause of mortality among SCD patients and the current therapy for ACS remains primarily supportive. Although flu typically causes a self-resolving upper respiratory track inflammation, it can progress into a life-threatening ALI in SCD patients. However, the mechanisms underlying flu severity in SCD remains unknown. Methods: We have developed a novel model of A/PR/8/34 (H1N1) influenza A virus (IAV)-induced respiratory infection in knock-in, humanized Townes SCD mice. Townes SCD (SS) and control (AS) mice were inoculated intranasally with a mild dose of IAV and lung injury was assessed over 14 days post-infection using lung histological scoring, oxygen saturation and body weight drop. Quantitative fluorescence intravital lung microscopy (qFILM) was performed using a multi-photon-excitation microscope at day 8, 10, 12 and 14 post-infection to assess thrombo-inflammation and vascular leakage in the lungs. Viral titer in lungs was assessed based on mRNA expression of viral M1 protein. Platelets were isolated for the biochemical assessment of the activation of antiviral signaling pathways. Results: SCD+Flu mice manifested significantly higher drop in oxygen saturation (<90%), body weight loss (≥ 20%) and development of hemorrhage, vascular congestion and edema (based on histology) in the lung than control+Flu mice, suggestive of the development of severe ALI in SCD+Flu than control+Flu mice. Surprisingly, although ALI and viral titer in the lung was completely resolved in control+Flu mice by day 12 post infection, SCD+Flu mice continued to manifest ALI and presence of IAV in the lung. Intravital microscopy revealed that the impaired resolution of ALI in SCD+Flu mice at day 12 was associated with lung microvasculature occlusion by neutrophil-platelets aggregates (NPAs), resulting in pulmonary ischemia and loss of blood-air barrier. In contrast to SCD mice, neutrophil-platelet aggregates were absent in the lung microcirculation of control+Flu mice at day 12 post infection. Western blot and co-immunoprecipitation analysis revealed assembly of viral-RNA sensing RIG-I/MAVS complex in SCD+Flu but not control+Flu mice platelets, suggestive of the activation of platelet-dependent anti-viral response in SCD+Flu mice. Conclusions: These findings suggest, for the first time, a role for platelet-dependent antiviral RIG-I signaling in promoting severe lung injury following flu infection in SCD. Currently, studies are in progress to identify the signaling pathways downstream of platelet RIG-I/MAVS that contribute to flu induced severe lung injury in SCD mice.

Neutrophil-TLR9 is a therapeutic target for acute chest syndrome in sickle cell disease

Circulating Extracellular Vesicles Enable IFN-I Response in Neutrophils of Sickle Cell Disease Mice

CD39 As a Master Regulator of Pulmonary Thrombosis in Sickle Cell Disease

Plasma NTPDase1 Activity Regulates Platelet Purinergic Signaling in Sickle Cell Disease

Circulating Neutrophil Extracellular Traps in the Pathogenesis of Acute Chest Syndrome of Sickle Cell Disease

Platelet-Neutrophil Aggregates Promote Pulmonary Arteriole Microembolism in Sickle Cell Disease

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

DNA Sensing By Platelets Promotes Acute Chest Syndrome in Sickle Cell Disease

Imatinib Protects Against Hypoxia/Reoxygenation Induced Lung and Kidney Injury in a Humanized Mouse Model for SCD

To the editor: Sickle cell disease (SCD) is associated with both microvascular and macrovascular complications.1 Stroke is considered 1 of the most common and disabling vascular complications in patients with SCD, occurring in approximately 24% of individuals by age 45.2,3 The etiology of stroke remains unclear, although preexisting obstructive lesions in major intracranial vessels have been reported.4 The microvasculature in SCD may be less reliable in maintaining viability in stroke border zones because of predisposition to microvascular occlusions by sickled red blood cells (RBCs). Patency of the microvessels may be particularly important in the setting of acute stroke in which tissues distal to the occlusion are hypoxic; this may trigger occlusive microvascular sickling. The effect of stroke size following macrovascular occlusion in SCD has not been previously reported to our knowledge. The effect of SCD on stroke size in humans is difficult to determine because there is no suitable control group for the SCD-related strokes that occur at relatively early ages.5 In this study, we sought to determine whether stroke size following macrovascular occlusion would be affected in mice with SCD. Male SCD and control mice were generated by bone marrow transplantation as previously described.6,7 SCD mice were anemic (hemoglobin: 9.7 ± 0.3 vs 12.6 ± 0.5 g/dL, P < .001) with splenomegaly (494.5 ± 51.5 vs 131 ± 11.5 mg, P < .001) compared with wild-type (WT) mice. Eight weeks after bone marrow transplantation, middle cerebral artery (MCA) occlusion was induced by photochemical injury as previously described.8 As early as 1 hour after occlusion, cortical discoloration representing the ischemic zone was greater in SCD mice compared with WT mice (n = 5/group) (Figure 1A-C). Another group of control and SCD mice (n = 10/group) were analyzed at day 3 following MCA occlusion to determine effects of SCD on completed stroke size as previously described.9 Although there was no mortality in the control group, mortality occurred in 4 SCD (P < .001) mice on days 2 (n = 2) and 3 (n = 2) following induction of stroke. Of the surviving mice, completed stroke size was greater in SCD mice compared with WT mice (Figure 1D-F). This was associated with widespread occlusion of the microvasculature by sickled RBCs in the stroke border zone (Figure 1G). Figure 1 Effect of SCD on stroke after MCA occlusion. At 1 hour following MCA occlusion, the area of erythema representing ischemic zone was greater in SCD compared with WT mice (A-C). At 3 days following MCA occlusion, infarct size by 2,3,5-triphenyltetrazolium ... Because the microvasculature may preserve the viability of the stroke border zone, it is likely that this was the cause of the increased stroke size. The increased mortality in SCD mice was unexpected and may be due to the triggering of a sickle cell crisis following induction of stroke. However, circulating sickled RBCs constituted only 1.1 ± 0.2% of the circulating RBCs following stroke. Overall, these findings indicate that in addition to being at high risk of stroke, SCD patients may suffer greater damage when stroke occurs. Although identification of the predisposing factors for macrovascular occlusion is the highest priority for stroke prevention in SCD, cellular events in the microvasculature could affect outcomes once macrovascular flow disturbance occurs. Therapies targeting microvascular sickling may reduce morbidity associated with stroke in SCD.

IFN-I Promotes T Cell-Independent Immunity and RBC Autoantibodies Via Modulation of B-1 Cell Subsets in Murine SCD

A Novel Hemoglobin-Binding Agent Reduces Plasma Free Hemoglobin and Partially Improves Vascular Function In Murine Hemolytic Anemia

Neutrophil Extracellular Traps Promote Lung Arteriole Occlusion in Sickle Cell Disease