Single-cell transcriptomic profiling of chronic obstructive pulmonary disease (COPD) lungs identified QKI, an RNA-binding protein, as a candidate emphysema-associated gene, but its epithelial role in COPD pathobiology remains unclear. We show that QKI expression is reduced in human COPD lungs and that alveolar type 2 epithelial (AT2) cell QKI protein levels correlate strongly with spirometric indices and diffusing capacity (DL CO ). Lung epithelium-specific QKI knockout mice (QKI Δ/Δ ) developed spontaneous airspace enlargement with emphysema-like mechanics, and QKI-deficient AT2 cells showed impaired spheroid colony formation and increased apoptosis. Integrated transcriptomic and proteomic analyses of primary AT2 cells revealed a selective reduction in functional mitochondrial (respiratory-chain and metabolic) protein abundance despite relatively preserved transcript levels, consistent with mitochondrial transcriptome-proteome discordance. QKI loss increased mtDNA abundance and TOMM20 staining but decreased ATP5A, indicating accumulation of structurally increased but functionally dysfunctional mitochondria. In human epithelial cells, CRISPR-mediated QKI deficiency reduced oxidative respiration, increased glycolytic reliance, elevated mitochondrial ROS and membrane potential, and increased apoptosis; these phenotypes were partially rescued by QKI re-expression. These findings identify epithelial QKI as a regulator of mitochondrial integrity and stress tolerance in COPD.

An early event after lung injury is extracellular matrix (ECM) remodeling and the formation of a provisional matrix. While megakaryocytes and platelets (Mgk/plt) play important roles in hemostasis, their impact on the matrix in lung injury is not fully understood. Using lung intravital microscopy, 3D large area scanning with multiphoton confocal hybrid imaging, and label-free quantitative proteomics, the matricellular protein thrombospondin-1 (TSP1) arising from Mgk/plt protects the lung from alveolar injury. Mgk/plt cell-specific Thbs1 knockout (cKO) mice show increased alveolar barrier disruption with exaggerated neutrophil-mediated injury. The cKO mice exhibit striking extracellular matrix re-organization with reduction in basement membrane matrix protein COL6A1 after injury. Moreover, cKO mice increase Mgk numbers to regions of fibrillar collagen deposition and alveolar leak. Our findings indicate Mgk/plt-derived TSP1 represents a key mechanism of matrix stabilization by protecting the basement membrane from neutrophil-mediated proteolytic damage, regulating injury severity and Mgk numbers at the alveolar interface.

Sickle cell disease (SCD) is the most common monogenic-hemolytic disorder affecting people of African ancestry. Adenosine diphosphate (ADP) released following intravascular hemolysis activates platelets by stimulating purinergic receptors to promote thrombosis. Despite brisk intravascular hemolysis, which releases high levels of ADP into plasma, and evidence of platelet and hemostatic activation, it remains elusive why only a subset of SCD patients develop lung thrombosis. Using real-time in vivo lung microscopy, we report a surprising finding that humanized SCD mice are protected from ADP-induced lung thrombosis, which is secondary to the degradation of ADP by CD39 present in circulating extracellular vesicles released by the lung endothelium. ADP-induced platelet aggregation is also impaired in the blood of SCD patients with elevated levels of CD39+ extracellular vesicles. CD39 polymorphism rs3176891A→G is associated with the incidence of lung thrombosis in SCD patients but not healthy humans of African ancestry. Remarkably, CD39+ extracellular vesicles are fewer and ADP-induced platelet aggregation is higher in the blood of SCD patients with rs3176891G allele. This study identifies a novel extracellular vesicle-dependent mechanism preventing lung thrombosis in SCD and reveals how CD39 polymorphisms may impair this protection to increase the risk for lung thrombosis in a subset of SCD patients. It remains unknown why only some sickle cell disease (SCD) patients develop lung thrombosis. Here, the authors show that an extracellular vesicle-dependent mechanism prevents lung thrombosis in SCD and how a CD39 polymorphism impairs this protection to promote lung thrombosis in subset of patients.

Pain constitutes a significant comorbidity associated with sickle cell disease (SCD). Analgesics serve as the primary method for pain management; however, the long-term effects of these drugs on the liver of SCD patients remain not completely understood. Using real-time intravital imaging, we analyzed the effect of non-steroidal analgesics (NSA) in the liver of control and SS (SCD) mice. Remarkably, we found completely opposing effects in the liver of control and SS mice post-NSA treatment. Whereas SS mice were able to better tolerate the NSA treatment acutely compared to their littermate controls, in the long term, these mice showed delayed resolution of liver injury and exacerbated fibrosis compared to control mice. Mechanistically, we found that SS mice were protected from cytotoxicity caused by NSA at baseline due to the significant activation of hepatic Kupffer cells, which produced heme-oxygenase 1 (HO-1). HO-1 promoted the activation of the cytoprotective enzyme Cyp3A11, which inhibited hepatic damage caused by NSA. However, in the long term, depletion of hepatic Kupffer cells led to reduced expression of HO-1, which blocked the activation of Cyp3A11, resulting in fibrosis and a delay in the resolution of liver injury and inflammation. These preclinical data provide a strong proof-of-concept for HO-1 as well as Cyp3A11 as cytoprotectors against NSA-induced liver damage in the Townes model of SCD and support further development of these compounds as potential novel therapies for end-organ damage in SCD.

Intrahepatic accumulation of cell-free hemoglobin (Hb) is a significant pathology linked with hemolytic disorders such as sickle cell disease (SCD). In addition to hepatic Kupffer cells, liver sinusoidal endothelial cells (LSECs) were recently reported to contribute to Hb clearance in SCD mice and patients via currently unknown endocytic mechanism. Using small molecule inhibitors of endocytic pathway components in primary human and mouse LSECs, we show that sickle-Hb (HbS) uptake by LSECs occurs predominantly through micropinocytosis or fluid-phase endocytosis. However, inhibiting clathrin-mediated endocytosis, receptor recycling, or drop in pH also significantly attenuated HbS uptake by LSECs. LSEC-driven HbS uptake was independent of haptoglobin. Finally, we found that presence of lipid droplets promotes endothelial HbS internalization while hypolipidemic condition inhibits it. In conclusion, the current study identifies previously unknown alternate mechanism of LSEC-mediated HbS internalization. Our findings also inform the need to evaluate the therapeutic potential of blocking these mechanisms to ameliorate hemolysis-associated liver damage in SCD and other hemolytic disorders.

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.

Acute painful vaso-occlusive episodes (VOEs) are the primary reason for emergency department visit by sickle cell disease (SCD) patients and contribute to significant morbidity in SCD. Crizanlizumab, a first-in-class humanized anti-P-selectin IgG2 monoclonal antibody, is approved in more than 40 countries for prevention of VOEs in 16 years or older SCD patients. Inclacumab, a fully human anti-P-selectin IgG4 mAb in clinical development is believed to have stronger affinity to P-selectin and greater maximal inhibition of cell-cell interactions than crizanlizumab. Using in vitro blinded experiments, we investigated whether crizanlizumab and inclacumab can be differentiated in terms of P-selectin binding affinity and inhibition of P-selectin-mediated cell adhesion in blood samples from healthy volunteers or SCD patients. Surface Plasmon Resonance revealed that inclacumab had higher P-selectin binding affinity than crizanlizumab, however, the inhibition of P-selectin-mediated cell adhesion was higher or comparable with crizanlizumab than inclacumab. Crizanlizumab and inclacumab were comparable in inhibiting leukocyte and erythrocyte adhesion to P-selection under vascular mimetic flow in microfluidic channels, leukocyte-platelet aggregation, and platelet aggregation in SCD patient or control human blood. In summary, these results suggest that comparable or higher inhibition of cell adhesion with crizanlizumab vs inclacumab does not correlate with P-selectin binding affinity. Ultimately, clinical trials are required to evaluate how crizanlizumab vs inclacumab translate into treatment outcomes in SCD patients.

Background Sickle cell disease (SCD) is the most common monogenic blood disorder that is widely recognized for its hallmark vaso-occlusive episodes (VOEs). VOEs are not only debilitatingly painful but also cause lasting damage to tissues and organs due to the repeated ischemic injury, emphasizing the need for an acute disease-modifying therapeutic to alleviate VOE burden. Treatment options for SCD are very limited and there remains a major gap for resolving acute VOE. We are therefore developing IHP-102 as an acute rescue therapeutic that targets the multifaceted pathomechanisms underlying VOE. IHP-102 is a novel glycan therapeutic that was designed to have polypharmacologic activity against complement and the vascular adhesion processes, both of which are recognized in the pathogenesis of VOE. The complex mechanisms involved in VOE demand robust intervention and a single therapeutic agent that targets multiple pathways offers great promise. Aims IHP-102 was previously shown to reduce vaso-occlusion by more than 80% in Townes SS mice, while historically P-selectin blockade reduced vaso-occlusions by only 50%. Here we aimed to extend these findings and further elucidate the contribution of complement activity of IHP-102 on vaso-occlusion. Methods We investigated IHP-102 in Townes SS (SCD) mice intravenously (IV) challenged with oxy-hemoglobin (IV oxyHb) to induce vaso-occlusion, which was quantified in the lung vasculature using quantitative fluorescence intravital lung microscopy (qFILM). Mice were treated with subcutaneous (SC) IHP-102 prior to IV oxy-Hb challenge. To determine the contribution of complement inhibition, we assessed the effect of IHP-102 on lung vaso-occlusion in SCD mice with P-selectin inhibited by either an anti-P-selectin function blocking Ab (RB40.34) or using SCD mice genetically deficient in P-selectin (SCD-Selp-/-). Results In Townes SCD-Selp-/- mice, IHP-102 reduced lung vaso-occlusion by more than 60% as measured by the number of pulmonary vaso-occlusions occlusions per field of view (#PVO/FOV) (n = 5 mice/group; p < 0.01). IHP-102 also reduced lung vaso-occlusion in SCD mice pretreated with P-selectin blocking Ab. These results indicate the anti-complement activity of IHP-102 provides significant additional therapeutic benefit beyond P-selectin inhibition. Summary/Conclusion While adhesion mechanisms through P-selectin are partially responsible for VOE, recent clinical data suggest targeting P-selectin alone may be insufficient. In contrast, the polypharmacologic activity of IHP-102 demonstrates strong therapeutic potential with profound benefit beyond P-selectin inhibition. Additionally, IHP-102 is dosed by SC route and can therefore be self-administered in the ambulatory setting, thus circumventing a largely ill-equipped healthcare system that has plagued SCD care. IHP-102 has the potential to transform management of VOE, offering significant relief from disease and the associated debilitating pain and organ damage.

In situ pulmonary arterial thrombosis (iPAT), occurring without concurrent deep vein thrombosis, is a life-threatening complication associated with various pathological conditions. The etiological mechanisms underlying iPAT remain poorly understood. Several studies suggest that circulating tissue factor (cTF) may contribute to the development of thrombotic complications; however, there is no direct in vivo evidence supporting the role of cTF in the pathogenesis of iPAT. Furthermore, although in vitro studies suggest that platelet anionic phospholipids enable cTF-initiated coagulation, how platelets contribute to cTF-dependent iPAT in vivo remains unknown. In the current study, we used quantitative fluorescence intravital lung microscopy to investigate the development of cTF-induced iPAT in live mice following intravascular administration of thromboplastin. To dissect the interplay between coagulation and platelet procoagulant activity, we assessed the effects of coagulation and platelet inhibition on iPAT development in vivo. Additionally, we conducted an in vitro clotting time assay using mouse plasma samples. Thromboplastin triggered iPAT in mice in a dose-dependent manner. IPAT involved the formation of platelet-rich thrombi at the bottle-neck junctions of pulmonary arterioles and capillaries, which was prevented by heparin. Notably, pretreatment of mice with annexin A5 or eptifibatide also completely abrogated thromboplastin-induced iPAT. These intravital microscopy findings were further corroborated by the in vitro clotting time assay. Our study provides in vivo evidence that cTF contributes to the development of iPAT. We demonstrate that the prothrombotic effect of cTF is dependent on platelet-αIIbβ3 signaling, which enhances platelet procoagulant activity, leading to accelerated coagulation and development of iPAT.

Sickle cell disease (SCD) causes debilitating chronic pain in over 50% of adults, highlighting the urgent need for new non-opioid-based therapies. Inflammation is central to SCD and is driven by activated neutrophils producing damage-associated molecular patterns (DAMPs), including neutrophil extracellular traps (NETs) via caspase-dependent gasdermin-D activation. We reported that neutrophil activation could be inhibited by interleukin-27 (IL-27), an anti-inflammatory cytokine shown to have the potential to regulate neuroinflammation (Alagbe et al. 2017). Therefore, we hypothesized that IL-27 could reduce chronic pain in SCD by inhibiting NET formation and neuroinflammation.MethodsWe analyzed stored plasma from adult patients with SCD in steady state and controls enrolled in an IRB-approved clinical trial. Patient were administered the Brief Pain Inventory short form for the assessment of pain severity (O'Brien et al. 2024). We quantified circulating NET levels as triple positive for citrullinated histones, extracellular DNA, and neutrophil elastase using imaging flow cytometry, and explored correlations with the patients' BPI scores. Additionally, fresh blood samples from prospectively enrolled SCD patients in steady state were treated ex vivo with hemin (20µM) ± recombinant human (rh) IL-27 (200 ng/mL) to assess neutrophil inflammatory signaling. Finally, aged Townes SS mice were treated with recombinant mouse (rm) IL-27 (IP 16 mg/Kg body weight in 200 μL saline) or sham for two weeks (3 doses/week); tactile, cold, and heat hypersensitivity were assessed at baseline and weekly. Circulating NETs were quantified at baseline and post-treatment, and circulating neutrophils and dorsal root ganglia (DRGs) were isolated and analyzed for inflammatory signaling and histology, respectively.ResultsThe study included 53 individuals with sickle cell disease (HbSS, HbSC, HbSβ⁺, and HbSβ⁰) and 17 age-matched healthy Black controls (mean age 38.6 ± 12.4 vs. 38.8 ± 10.6 years). Despite being in steady state, a majority of the SCD patients reported pain in the prior 24 hours. Patients also had higher levels of circulating NETs compared to the healthy controls (p = 0.042), and their pain scores positively correlated with circulating NET levels (r = 0.32) and inversely with plasma IL-27 levels (r = - 0.33). Hemin induced caspase-5-dependent GSDMD activation in SCD neutrophils ex vivo, which was inhibited by rhIL-27.Townes SS mice (n = 6) treated with rmIL-27 showed significantly reduced heat and cold hypersensitivity (p < 0.01 for both), with no change in tactile sensitivity compared to sham (n = 5). The rmIL-27-treated mice had a reduction in the circulating NET levels, while the sham mice had an increase in circulating NETs compared to their respective baseline levels (p = 0.08). IL-27 treatment also tended to reduce caspase-5-dependent GSDMD activation in neutrophils, and H&E staining revealed decreased inflammatory cell infiltration in the DRG compared to controls.ConclusionsThese findings suggest that IL-27 may attenuate chronic pain in SCD by suppressing neutrophil activation, NET formation, and neuroinflammation. The observed correlations between pain, circulating NETs, and IL-27 levels in patients, along with the therapeutic effects of IL-27 in a preclinical SCD model, support its potential as a novel, non-opioid therapeutic strategy for SCD-associated pain. Further studies are warranted to explore IL-27–based interventions in clinical settings.

Introduction Granulocyte-colony stimulating factor (G-CSF) administration is known to induce severe vaso-occlusive crisis (VOC) in patients with sickle cell disease (SCD). G-CSF and neutrophils rise acutely during VOC, whereas an elevated circulating neutrophil count at steady state is a prognostic factor of SCD severity. G-CSF is induced by many inflammatory triggers, including lipopolysaccharides (LPS), tumor necrosis factor alpha (TNFα) and toll-like receptor 4 activation, which provoke VOC in SCD mice. Importantly, G-CSF induces neutrophil extracellular traps (NETs) formation and activates platelets and monocytes, leading to endothelial cell activation and thrombo-inflammation in SCD. We report the first preclinical evidence validating G-CSF receptor (G-CSFR) blockade as a therapeutic target to prevent VOC in SCD. Methods Townes knock-in humanized SCD mice were exposed to hypoxia-reoxygenation (HR) or intravenously (IV) challenged with oxy-hemoglobin (oxy-Hb), LPS, or G-CSF to induce VOC. Some mice were pretreated IV with VR81, an anti-mouse G-CSFR monoclonal antibody (mAb), prior to VOC induction. Blood flow and NETs formation were assessed by intravital microscopy of the skin and lungs. Liver and lung tissues were examined for inflammation by Western blots and immunofluorescence staining, respectively. Gene expression analyses were performed on transcriptomic datasets from the blood of SCD patients with/without VOC and healthy humans with/without treatment with G-CSF and/or CSL324, an anti-human G-CSFR mAb under development. Results A single IV dose of VR81 in mice had a half-life of ~7 days. Full receptor occupancy in neutrophils was immediate, maintained for at least 1 week, and confirmed by the complete inhibition of phosphorylated STAT3, the signal transduction mediator of G-CSFR signaling. G-CSF administered IV to SCD mice dose-dependently induced microvascular vaso-occlusion in the skin. Vaso-occlusion in the skin was prevented by VR81 administration IV 1 hour or 7 days prior to VOC induction with G-CSF, HR, or oxy-Hb. Importantly, VR81 resolved skin vaso-occlusion in SCD mice when administered 40 minutes after VOC induction. Biomarker analysis revealed several effects caused by G-CSFR blockade. In the liver, expression of pro-inflammatory endothelial adhesion molecules VCAM-1, ICAM-1, and E-selectin and nuclear factor-ĸB-phospho-p65 were markedly decreased, while expression of anti-inflammatory heme oxygenase-1 and nuclear localization of nuclear factor erythroid 2-related factor 2 were increased. Intravital microscopy in the lungs revealed that VR81 pretreatment also prevented neutrophil-platelet aggregate-dependent lung vaso-occlusion in oxy-Hb challenged SCD mice, with a reduced number of NETs within the lung and blood circulation. In the lungs, the number of neutrophils, the expression of pro-adhesive Weibel-Palade body P-selectin, and von Willebrand factor were decreased. Long-term administration of VR81 once per week for 5 months reduced signs of chronic organ injury. The weight of the right ventricle was normalized, suggesting a possible reduction in pulmonary hypertension. The white blood cell count was also reduced. Transcriptomic analyses support the translatability of G-CSFR blockade to SCD patients. We previously published a gene signature of differentially expressed genes in blood after G-CSF administration to healthy humans. In SCD patients, this G-CSF gene signature was significantly enriched during VOC vs. baseline (p-value < 10-3) when analyzed using microarray and RNA-Seq datasets. Conclusions Literature and our data strongly support the critical role of G-CSF in thrombo-inflammatory pathobiology in VOC. Because G-CSF activates multiple inflammatory pathways that trigger vaso-occlusion, G-CSFR is a suitable target for drug intervention to prevent VOC. Our data suggest multiple anti-inflammatory effects in SCD, including prevention of NET formation, which provide a plausible mechanism of action for G-CSFR blockade. The known biology of NETs aligns with a role for G-CSFR in SCD. The NETs formation and their rise during VOC have been confirmed from the literature for patients with SCD. CSL324 was safe and well tolerated in repeat-dose toxicological studies in non-human primates and has completed a phase 1 trial, with satisfactory safety (some risk of transient neutropenia) and pharmacokinetic profiles. A phase 2 trial in patients with SCD will seek clinical proof of concept for VOC prevention.

Intrahepatic accumulation of cell-free hemoglobin (Hb) is a significant pathology linked with hemolytic disorders such as sickle cell disease (SCD). In addition to hepatic Kupffer cells, liver sinusoidal endothelial cells (LSECs) were recently reported to contribute to Hb clearance in SCD mice and patients via currently unknown endocytic mechanism. Using small-molecule inhibitors of endocytic pathway components in primary human and mouse LSECs, we show that sickle-Hb (HbS) uptake by LSECs occurs predominantly through micropinocytosis or fluid-phase endocytosis. However, inhibiting clathrin-mediated endocytosis, receptor recycling, or drop in pH also significantly attenuated HbS uptake by LSECs. LSEC-driven HbS uptake was independent of haptoglobin. Finally, we found that the presence of lipid droplets promotes endothelial HbS internalization, whereas hypolipidemic condition inhibits it. In conclusion, this study identifies previously unknown alternative mechanism of LSEC-mediated HbS internalization. Our findings also inform the need to evaluate the therapeutic potential of blocking these mechanisms to ameliorate hemolysis-associated liver damage in SCD and other hemolytic disorders.