Prithu Sundd

Patients with severe hemophilia A suffer from recurrent bleeding in synovial joints, which leads to development of hemophilic arthropathy, a progressive condition characterized by permanent joint damage, chronic pain and poor quality of life. Current treatment with factor VIII replacement or factor VIII mimetic therapy provides only partial protection from joint bleeding. Unfortunately, targeted therapies to prevent the development of hemophilic arthropathy remain an unmet clinical need, and the etiological mechanism underlying joint damage in hemophilia is still poorly understood. Using the experimental model of hemophilic arthropathy in Factor 8 total knockout (F8TKO) mice and blood samples from hemophilia A patients with or without active joint bleeding, we show that acute joint bleeding promotes an inflammatory milieu in hemophilia, which triggers the type-I interferon response and downstream signaling in neutrophils, leading to systemic shedding of neutrophil extracellular traps (NETs). Remarkably, therapeutic inhibition of type-I interferon receptor attenuates shedding of NETs and reduces the severity of joint damage in F8TKO mice. The current study is the first to identify a role for NETs in the pathogenesis of hemophilic arthropathy and demonstrates the therapeutic potential of targeting neutrophil-dependent type-I interferon signaling to attenuate the progression of joint damage in hemophilia.

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.

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.

Cigarette smoking is associated with a higher risk of ICU admissions among patients with flu. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice preexposed to CS but not room air for 4 weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS- and flu-exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest, for the first time to our knowledge, that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS-induced severe flu.