Biology subjects

Hekman, R. M.

Publications and source records attributed to Hekman, R. M..

4 recordsLinked to original sources

bioRxiv2021

Platelet proteome analysis reveals an early hyperactive phenotype in SARS-CoV-2-infected humanized ACE2 mice

Coronavirus disease-2019 (COVID-19) provokes a hypercoagulable state with increased incidence of thromboembolism and mortality. Platelets are major effectors of thrombosis and hemostasis. Suitable animal models are needed to better understand COVID-19-associated coagulopathy (CAC) and underlying platelet phenotypes. Here, we assessed K18-hACE2 mice undergoing a standardized SARS-CoV-2 infection protocol to study dynamic platelet responses via mass spectrometry-based proteomics. In total, we found significant changes in >1,200 proteins. Strikingly, protein alterations occurred rapidly by 2 days post-infection (dpi) and preceded outward clinical signs of severe disease. Pathway enrichment analysis of 2dpi platelet proteomes revealed that SARS-CoV-2 infection upregulated complement-coagulation networks (F2, F12, CFH, CD55/CD59), platelet activation-adhesion-degranulation proteins (PF4, SELP, PECAM1, HRG, PLG, vWF), and chemokines (CCL8, CXCL5, CXCL12). When mice started to lose weight at 4dpi, pattern recognition receptor signaling (RIG-I/MDA5, CASP8, MAPK3), and interferon pathways (IFIT1/IFIT3, STAT1) were predominant. Interestingly, SARS-CoV-2 spike protein in the lungs was observed by immunohistochemistry, but in platelets was undetected by proteomics. Similar to patients, K18-hACE2 mice during SARS-CoV-2 infection developed progressive lymphohistiocytic interstitial pneumonia with platelet aggregates in the lungs and kidneys. In conclusion, this model recapitulates activation of coagulation, complement, and interferon responses in circulating platelets, providing valuable insight into platelet pathology during COVID-19. Key PointsO_LISARS-CoV-2-infected humanized ACE2 mice recapitulate platelet reprogramming towards activation-degranulation-aggregation. C_LIO_LIComplement/coagulation pathways are dominant in platelets at 2 days post-infection (dpi), while interferon signaling is dominant at 4dpi. C_LI

immunology↗

bioRxiv2021

Recombinant Lloviu virus as a model to study inaccessible zoonotic viruses

Next generation sequencing has revealed the presence of many RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, many of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Recombinant LLOV (rLLOV) displays typical filovirus features, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal against pandemic preparedness.

microbiology↗

bioRxiv2021

Macrophages govern antiviral responses in human lung tissues protected from SARS-CoV-2 infection

The majority of SARS-CoV-2 infections among healthy individuals result in asymptomatic to mild disease. However, the immunological mechanisms defining effective lung tissue protection from SARS-CoV-2 infection remain elusive. Unlike mice solely engrafted with human fetal lung xenograft (fLX), mice co-engrafted with fLX and a myeloid-enhanced human immune system (HNFL mice) are protected against SARS-CoV-2 infection, severe inflammation, and histopathology. Effective control of viral infection in HNFL mice associated with significant macrophage infiltration, and the induction of a potent macrophage-mediated interferon response. The pronounced upregulation of the USP18-ISG15 axis (a negative regulator of IFN responses), by macrophages was unique to HNFL mice and represented a prominent correlate of reduced inflammation and histopathology. Altogether, our work shed light on unique cellular and molecular correlates of lung tissue protection during SARS-CoV-2 infection, and underscores macrophage IFN responses as prime targets for developing immunotherapies against coronavirus respiratory diseases. HIGHLIGHTSO_LIMice engrafted with human fetal lung xenografts (fLX-mice) are highly susceptible to SARS-CoV-2. C_LIO_LICo-engraftment with a human myeloid-enriched immune system protected fLX-mice against infection. C_LIO_LITissue protection was defined by a potent and well-balanced antiviral response mediated by infiltrating macrophages. C_LIO_LIProtective IFN response was dominated by the upregulation of the USP18-ISG15 axis. C_LI

microbiology↗

bioRxiv2020

Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease

The incompletely understood pathogenesis of pulmonary fibrosis (PF) and lack of reliable preclinical disease models have limited development of effective therapies. An emerging literature now implicates alveolar epithelial type 2 cell (AEC2) dysfunction as an initiating pathogenic event in the onset of a variety of PF syndromes, including adult idiopathic pulmonary fibrosis (IPF) and childhood interstitial lung disease (chILD). However, inability to access primary AEC2s from patients, particularly at early disease stages, has impeded identification of disease-initiating mechanisms. Here we present an in vitro reductionist model system that permits investigation of epithelial-intrinsic events that lead to AEC2 dysfunction over time using patient-derived cells that carry a disease-associated variant, SFTPCI73T, known to be expressed solely in AEC2s. After generating patient-specific induced pluripotent stem cells (iPSCs) and engineering their gene-edited (corrected) counterparts, we employ directed differentiation to produce pure populations of syngeneic corrected and mutant AEC2s, which we expand >1015 fold in vitro, providing a renewable source of cells for modeling disease onset. We find that mutant iPSC-derived AEC2s (iAEC2s) accumulate large amounts of misprocessed pro-SFTPC protein which mistrafficks to the plasma membrane, similar to changes observed in vivo in the donor patients AEC2s. These changes result in marked reduction in AEC2 progenitor capacity and several downstream perturbations in AEC2 proteostatic and bioenergetic programs, including a late block in autophagic flux, accumulation of dysfunctional mitochondria with consequent time-dependent metabolic reprograming from oxidative phosphorylation to glycolysis, and activation of an NF-{kappa}B dependent inflammatory response. Treatment of SFTPCI73T expressing iAEC2s with hydroxychloroquine, a medication commonly prescribed to these patients, results in aggravation of autophagy perturbations and metabolic reprogramming. Thus, iAEC2s provide a patientspecific preclinical platform for modeling the intrinsic epithelial dysfunction associated with the inception of interstitial lung disease.

cell biology↗