Biology subjects

Doerflinger, M.

Publications and source records attributed to Doerflinger, M..

7 recordsLinked to original sources

bioRxiv2026

IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19.

Vascular dysfunction and coagulopathy are hallmarks of severe COVID-19. How SARS-CoV-2 infection drives endothelial dysfunction, despite the virus not infecting or replicating in endothelial cells, remains controversial. Here, we used an in vitro co-culture model of the human pulmonary epithelial-endothelial cell barrier to investigate which inflammatory mediators drive endothelial dysfunction during SARS-CoV-2 infection. SARS-CoV-2 infection of primary human bronchial epithelial cells increased adjacent endothelial cell expression of the leukocyte adhesion marker ICAM-1, disrupted endothelial VE-cadherin junctions, promoted endothelial cell death, and promoted platelet adherence to gaps in the endothelial monolayers. Dexamethasone treatment rescued these dysregulated endothelial phenotypes in infected co-cultures, confirming that inflammatory signalling was the primary driver of SARS-CoV-2-induced endothelial dysfunction. Specifically, epithelial-derived TNF and IL-1{beta} promoted endothelial dysfunction, as inhibition of TNF or IL-1R signalling blocked SARS-CoV-2-induced endothelial dysfunction in co-cultures. SARS-CoV-2-infected wild-type mice, but not TNF, IL-1{beta}, or TNF/IL-1{beta}- deficient mice, displayed increased endothelial ICAM-1 expression, while an anti-IL-1{beta} monoclonal antibody prevented SARS-CoV-2-induced ICAM-1 expression and fibrin deposition in aged K18-ACE2 mice. Our data indicate that TNF and IL-1{beta} are the specific cytokines that drive multiple aspects of endothelial dysfunction during acute SARS-CoV-2 infection, and that inhibiting their signalling pathways may provide therapeutic benefit in preventing vascular complications of COVID-19.

cell biology↗

bioRxiv2025

Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

microbiology↗

bioRxiv2025

Lipid nanoparticle delivered intrabodies for inhibiting necroptosis and pyroptosis

Intrabodies are intracellularly expressed high-affinity protein binders such as nanobodies and monobodies that offer an alternative approach to small molecules. However, the maturation of intrabody technology into new therapeutic modalities has been limited by the availability of a clinically relevant delivery system enabling sufficiently high levels of protein to be expressed in the cytosol. Here, we use lipid nanoparticle (LNP) systems based on clinically approved formulations for the efficient intracellular delivery of mRNAs encoding for intrabodies targeting mixed lineage kinase domain-like pseudokinase (MLKL) and apoptosis-associated speck-like protein containing a CARD (ASC), key mediators of the necrotic cell death modalities, necroptosis and pyroptosis, respectively. LNP delivery of intrabody mRNA resulted in robust protein expression, with a MLKL binding intrabody preventing MLKL membrane translocation and protecting against necroptotic cell death. Similarly, LNP delivery of a bivalent intrabody targeting the inflammasome adaptor protein ASC protected against NLRP3 and AIM2 inflammasome-driven responses, including caspase-1 and IL-1{beta} activation and gasdermin D-driven pyroptotic killing. These findings establish that LNPs harbouring anti-necrotic intrabody mRNAs allow for sufficient intracellular expression to neutralize necrotic cell death signalling and provide a general, clinically relevant, strategy for delivering therapeutic intrabodies into cells.

bioengineering↗

bioRxiv2024

Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

microbiology↗

bioRxiv2023

Voltage-gated T-type calcium channel blockers reduce apoptotic body-mediated SARS-CoV-2 cell-to-cell spread and subsequent cytokine storm

SARS-CoV-2 typically utilises host angiotensin-converting enzyme 2 (ACE2) as a cellular surface receptor and host serine protease TMPRSS2 for the proteolytic activation of viral spike protein enabling viral entry. Although macrophages express low levels of ACE2, they are often found positive for SARS-CoV-2 in autopsied lungs from COVID-19 patients. As viral-induced macrophage inflammation and overwhelming cytokine release are key immunopathological events that drives exacerbated tissue damage in severe COVID-19 patients, insights into the entry of SARS-CoV-2 into macrophages are therefore critical to understand COVID-19 pathogenesis and devise novel COVID-19 therapies. Mounting evidence suggest that COVID-19 pathogenesis is associated with apoptosis, a type of programmed cell death that often leads to the release of numerous large extracellular vesicles (EVs) called apoptotic bodies (ApoBDs). Here, we showed that ApoBDs derived from SARS-CoV-2-infected cells carry viral antigens and infectious virions. Human monocyte-derived macrophages readily efferocytosed SARS-CoV-2-induced ApoBDs, resulting in SARS-CoV-2 entry and pro-inflammatory responses. To target this novel ApoBD-mediated viral entry process, we screened for ApoBD formation inhibitors and discovered that T-type voltage-gated calcium channel (T-channel) blockers can inhibit SARS-CoV-2-induced ApoBD formation. Mechanistically, T-channel blockers impaired the extracellular calcium influxes required for ApoBD biogenesis. Importantly, blockade of ApoBD formation by T-channel blockers were able to limit viral dissemination and virus-induced macrophage inflammation in vitro and in a pre-clinical mouse model of severe COVID-19. Our discovery of the ApoBD-efferocytosis-mediated viral entry reveals a novel route for SARS-CoV-2 infection and cytokine storm induction, expanding our understanding of COVID-19 pathogenesis and offering new therapeutic avenues for infectious diseases.

cell biology↗

bioRxiv2022

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. We show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-{beta} induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

immunology↗

bioRxiv2020

Mechanism and inhibition of SARS-CoV-2 PLpro

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications1,2. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses3. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.

biochemistry↗