Biology subjects

Larcher, T.

Publications and source records attributed to Larcher, T..

12 recordsLinked to original sources

bioRxiv2026

Intracellular accumulation of AAV6 genomes governs microglia reactivity and the inflammatory response in retinal gene therapy

Adeno-associated viral (AAV) vectors possess immense potential for gene therapy applications. However, microglia can mount an immune response against AAV vectors. Yet, microglia are therapeutic targets since they play a driving role in the progression of neurodegenerative diseases. Thus, it is critical to understand the cellular processes driving the microglia-mediated immune response against AAV vectors. To this end, we modelled AAV6 induced inflammation upon retinal gene therapy in minipigs. Upon AAV6 administration, the IBA1+ cells demonstrated a morphological transition to the amoeboid form, indicating microglial reactivity. We also observed the invasion of peripheral immune cells in the retina. To elucidate the underlying molecular mechanisms, we transduced human iPSC derived microglia-like cells (iMGLs) in vitro with AAV6 and AAV8 respectively. Only AAV6 transduced iMGLs exhibited morphological features and a transcriptomic signature of microglial reactivity. The magnitude of intracellular AAV6 viral genomes was significantly higher as compared to the AAV8 condition. The elevated levels of intracellular AAV6 genomes induced a DNA damage response in AAV6-transduced iMGLs, which is a key feature of neuroinflammation-linked microglial reactivity. Thus, our work highlights the impact of intracellular viral genome accumulation on microglial reactivity and its implications for retinal gene therapy associated inflammation.

neuroscience↗

bioRxiv2025

Nipah virus Malaysia and Bangladesh strain-induced pathogenesis in mice lacking type I interferon receptor signaling

Nipah virus (NiV) is a zoonotic highly pathogenic Paramyxovirus inducing lethal outbreaks of encephalitis and severe acute respiratory syndrome (SARS) with an average case-fatality rate of 75%. Two viral strains, NiV-Malaysia (NiV-Mal) and NiV-Bangladesh (NiV-Ban), associated to distinct geographical distribution, route of transmission, symptoms and lethality have been described. Due to the permanent threat of these emerging infections and the lack of approved therapeutics, it is crucial to improve our understanding regarding NiV-associated pathogenesis. Mice represent a small and accessible animal model, provided with numerous biological tools for the functional assessment of different genes related to antiviral response. Here, we analyze the susceptibility of mice deficient for type I interferon receptor (IFNAR KO) to infection with either NiV-Mal or NiV-Ban through intraperitoneal or intranasal routes. Our results show that IFNAR KO mice are susceptible to NiV-Ban infection via intraperitoneal route, although to a lesser extent than NiV-Mal, and develop encephalitis and pulmonary syndrome with viral propagation to different organs and lethal outcome in 60% of infected animals. In addition, intranasal administration of both viral strains led to a subclinical infection with viral replication in brain and lungs and production of virus-specific neutralizing antibodies. These results indicate IFNAR KO mice as a small animal model permitting comparative studies of the immunopathogenesis perpetrated by both NiV-Mal and NiV-Ban infections. Author summaryAvailability of small animal models represents a major issue to characterize virus-associated pathogenesis to further implement therapeutic strategies. Previous studies performed with NiV-Mal and NiV-Ban strains in wild-type (WT) mice did not indicate signs of NiV disease. However, when challenged with NiV-Mal, IFNAR KO mice suffered fatal outcomes, thus providing useful information on viral immunopathogenesis. Surprisingly, while being the more frequently re-emerging virus strain, no infection studies with NiV-Ban have been performed in IFNAR KO mice so far. Here, we sought comparing pathogenesis after NiV-Mal- and NiV-Ban-infection following IP and IN inoculations in IFNAR KO mice. Indeed, our results highlighted that contrary to fatal IP challenge, IN inoculation lead to a subclinical infection with both NiV strains. Moreover, we determined that NiV-Mal is more pathogenic than NiV-Ban following IP infection. Finally, distinct histopathological manifestations and tropism were associated with each viral strain and specific route of infection. Overall, our study implies that IFNAR KO mice represent a useful animal model to study strain-specific NiV pathophysiology.

microbiology↗

bioRxiv2025

The MARK2 kinase acts as a gatekeeper of CD28-dependent co-stimulation in T cells

Naive T cell activation requires not only antigen recognition through the TCR but also a co-stimulatory signal, mainly provided by CD28. Here, using a T cell-specific conditional knockout (cKO) model, we identify the microtubule-affinity kinase 2 (MARK2) as a key intracellular checkpoint that limits CD28-mediated co-stimulation. In vivo, MARK2 deficiency promotes the development of central memory T cells, enhances basal glycolysis activity in naive CD8 T cells, and leads to the development of systemic autoimmunity in aged mice. In MARK2-deficient CD8 T cells, TCR engagement alone drives sustained proliferation, cytokine production, and glycolysis, processes that normally require CD28 co-stimulation. Single-cell transcriptomic analysis reveals that MARK2 regulates the expression of genes involved in CD28 signaling and metabolic switch. We show that MARK2 restrains the PI3K-AKT-mTORC1 pathway by limiting CD28-driven transcriptional and metabolic programs. Mechanistically, we demonstrate that MARK2 phosphorylates CREB regulated transcription coactivator 2 (CRTC2) and suppresses CREB-mediated transcription and mTOR activation, whereas CD28 engagement lifts this inhibition. Together, our results redefine the role of CD28 that not only amplifies TCR signaling but also relieves a MARK2-dependent inhibitory signal. This work provides new insights into T cell activation, metabolism and immune tolerance with potential implications for immunotherapeutic strategies in cancer and autoimmunity.

immunology↗

bioRxiv2025

Flagellin aerosol administration improves the efficacy of antibiotic treatment in Actinobacillus pleuropneumoniae infected pigs

Swine infections with Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, lead to significant production losses. Surviving pigs may harbor antibiotic-resistant bacteria, highlighting the need for alternatives to reduce antimicrobial use. Stimulation of respiratory innate immunity via Toll-like receptor 5 (TLR5) agonists such as FLAMOD (a recombinant flagellin), has emerged as a promising strategy for combating bacterial lung infections. In a recent study, aerosolized administration of FLAMOD was found to elicit respiratory immune responses in pigs. This study assessed FLAMODs protective effects against App, either as a prophylactic intervention or as an adjunct to antibiotic therapy. Prophylactic administration of aerosolized FLAMOD via nebulization before App challenge failed to confer protection. However, in a therapeutic setting, where pigs were infected with App and treated with either a subtherapeutic dose of penicillin G (PenG) alone or a combination of PenG and aerosolized FLAMOD. Notably, the FLAMOD and PenG combination significantly improved bacterial clearance from the lungs. Moreover, FLAMOD-PenG treated pigs exhibited a trend toward increased weight gain and fewer lung lesions compared to those receiving PenG alone. These findings provide proof for the concept that FLAMOD enhances antibiotic efficacy, potentially lowering the required antibiotic dose and contributing to antimicrobial resistance mitigation in pigs.

immunology↗

bioRxiv2024

Conserved RNA-binding protein interactions mediate syntologous lncRNA functions

Syntologous long noncoding RNAs (lncRNAs) are loci with conserved genomic positions that often show little or no sequence similarity. Despite diverging primary sequences, lncRNA syntologs from distant species can carry out similar functions. However, determinants underlying conserved functions of syntologous lncRNA transcripts with no sequence similarity remain unknown. Using CASC15 and melanoma formation as a paradigm for fast evolving lncRNAs and their functions, we found that human and zebrafish CASC15 syntologs with no detectable sequence similarity retained their function across 450 million years of evolution. Similar to the casc15-deficient zebrafish, CASC15-mutant human melanoma cells show increased cell migration. Expression of human CASC15 in zebrafish rescues loss of casc15 function by attenuating melanoma formation. This conserved function is supported by a set of RNA-binding proteins, interacting with both zebrafish and human CASC15 transcripts. Together, our findings demonstrate that conserved RNA-protein interactions can define functions of rapidly evolving lncRNA transcripts.

molecular biology↗

bioRxiv2024

Advancing Liver Gene Therapy: Enhanced Transduction with GalNAc-Bioconjugated rAAV Capsids

This study investigates novel approaches to improve targeted gene delivery to the liver, a crucial organ for metabolic processes that faces vulnerabilities from various pathologies. Adeno-associated virus (AAV)-based gene therapy has emerged as a promising approach for liver targeting, with numerous investigational avenues. However, administration of high doses of AAV vectors present safety concerns, often requiring the use of corticosteroids and immunosuppression to mitigate immune adverse events. To address this, substantial efforts are underway to engineer optimized capsids to enhance the efficiency and specificity of recombinant AAV (rAAV) targeting hepatocytes, aiming to reduce required dosages. In this study, we employed bioconjugation chemistry to target the Asialoglycoprotein receptor (ASGPR), a C-type lectin abundantly expressed at the surface of hepatocyte membranes. We demonstrated that covalently attaching carbohydrates derived from GalNAc (a known ASGPR ligand) to lysine amino-acids on the rAAV2 capsid significantly enhanced in vivo liver transduction efficiency in mice. These optimized vectors present a promising avenue for the treatment of a spectrum of liver diseases, providing an alternative solution within the framework of liver gene therapy.

bioengineering↗

bioRxiv2024

The effects of iron deficient and high iron diets on SARS-CoV-2 lung infection and disease

The severity of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often dictated by a range of comorbidities. A considerable literature suggests iron deficiency and iron overload may contribute to increased infection, inflammation and disease severity, although direct causal relationships have been difficult to establish. Here we generate iron deficient and iron loaded C57BL/6J mice by feeding low and high iron diets, with mice on a normal iron diet representing controls. All mice were infected with a primary omicron XXB SARS-CoV-2 isolate and lung inflammatory responses were analyzed by histology, immunohistochemistry and RNA-Seq. Compared with controls, iron deficient mice showed no significant changes in lung viral loads or histopathology, whereas, iron loaded mice showed slightly, but significantly, reduced lung viral loads and histopathology. Transcriptional changes were modest, but illustrated widespread dysregulation of inflammation signatures for both iron deficient vs. controls, and iron loaded vs. controls. Some of these changes could be associated with detrimental outcomes, whereas others would be viewed as beneficial. Diet-associated iron deficiency or overload thus induced modest modulations of inflammatory signatures, but no significant histopathologically detectable disease exacerbations. Author summaryA diet deficient in iron can lead to anemia, a widespread problem worldwide. A diet with excessive iron is less common, but can be associated with excessive consumption of iron supplements. We investigate herein using a mouse model, whether low or high iron diets predispose to detrimental outcomes in the lungs after infection with SARS-CoV-2. A considerable literature suggests iron dysregulation would promote infection and inflammation. However, we found, although inflammatory responses showed modest modulations, viral loads were unaffected or slightly reduced, and lung histopathology was either unaffected or indicated slightly less severe disease. These findings do not support a view that low or high iron diets represent comorbidities predisposing to overt detrimental outcomes for acute COVID-19 lung disease.

pathology↗

bioRxiv2023

Microplastics dysregulate innate immunity in the SARS-CoV-2 infected lung

Global microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis. Herein we described the effects of azide-free, 1 {micro}m polystyrene MP beads co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum using a mouse model of mild COVID-19. Lung virus titres and viral RNA levels were not significantly affected by MPs, with overt clinical or histopathological changes also not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 days post infection (dpi) and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the cytokine release syndrome signature seen in some severe COVID-19 patients. This study adds to a growing body of literature suggesting that MPs can dysregulate inflammation in specific disease settings. Graphical Abstract HIGHLIGHTSO_LIA single inoculation of microplastics dysregulated SARS-CoV-2 lung inflammation C_LIO_LIAt the peak of SARS-CoV-2 infection microplastics decreased early innate responses C_LIO_LILater post infection microplastics promoted a "cytokine release syndrome" signature C_LIO_LIA key mechanism may involve the inhibition of the phagocytosis of infected cells C_LIO_LIAzide-free microplastics were used, with no elevated ROS responses identified C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/567745v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@73eefborg.highwire.dtl.DTLVardef@14eb683org.highwire.dtl.DTLVardef@b0638aorg.highwire.dtl.DTLVardef@571512_HPS_FORMAT_FIGEXP M_FIG Postulated mechanisms whereby microplastics might decrease the proinflammatory responses 2 days after SARS-CoV-2 infection, yet promote the proinflammatory cytokine release syndrome signature at 6 days post infection. C_FIG

microbiology↗

bioRxiv2023

Characterization of an Australian outbreak Japanese Encephalitis virus genotype 4 isolate in mice and human cortical brain organoids

BackgroundHuman infections with Japanese encephalitis virus (JEV) are a leading cause of viral encephalitis. An unprecedented outbreak of JEV genotype 4 was recently reported in Australia, with an isolate (JEVNSW/22) obtained from a stillborn piglet brain. MethodsHerein we compared the neuropathology of JEVNSW/22, JEVFU (genotype 2) and JEVNakayama (genotype 3) in adult C57BL/6J wild-type mice, mice deficient in interferon regulatory factor 7 (Irf7-/-), and mice deficient in type I interferon receptor (Ifnar-/-), as well as in human cortical brain organoids (hBOs). Using human serum post-Imojev vaccination, we performed neutralisation assays to determine JEVNSW/22 susceptibility to vaccine responses. FindingsIn C57BL/6J and Irf7-/- mice with lethal outcomes, brain infection and histopathological lesions recapitulated those seen in humans and primates. JEV was universally lethal in Ifnar-/- mice by day 3 with histological signs of brain hemorrhage, but produced no other detectable brain infection or lesions, with viral protein detected in blood vessels but not neurons. We thus describe a new Irf7-/- mouse model for JEVNSW/22, which had increased viremia compared to C57BL/6J mice, allowing for lethal neuroinvasive infection in one mouse. Overall, JEVNSW/22 was less neurovirulent than other JEV isolates in C57BL/6J and Irf7-/- mice, and was more sensitive to type I interferon. All JEV isolates showed robust cytopathic infection of human cortical brain organoids, albeit lower for JEVNSW/22. We also show that Imojev vaccination in humans induced neutralizing antibodies against JEVNSW/22, with the level of cross-neutralisation related to the conservation in envelope protein amino acid sequences for each isolate. InterpretationOur study establishes JEVNSW/22 mouse models of infection, allowing for possible lethal neuroinvasive infection that was rarer than for other JEV genotypes. JEV vaccination regimens may afford protection against this newly emerged JEV genotype 4 strain, although neutralizing antibody responses are sub-optimal. FundingQIMRB received a generous philanthropic donation from the Brazil Family Foundation awarded to D.J.R. to support Japanese Encephalitis virus research at QIMRB. A.S. holds an Investigator grant from the National Health and Medical Research Council (NHMRC) of Australia (APP1173880). We also acknowledge the intramural grant from QIMR Berghofer awarded to R.S. and D.J.R. for purchase of the CelVivo Clinostar incubator for producing human cortical brain organoids. The project "Japanese encephalitis vaccine via the intradermal route in children and adults (JEVID-2): A clinical trial comparing the immunogenicity and safety of Japanese encephalitis vaccine administered by subcutaneous and intradermal routes" being conducted by G.D., N.G., and N.W. was funded by the Sydney Childrens Hospitals Network and New South Wales Health. Research in context Evidence before the studyJEV from the historically rare genotype 4 recently emerged in Australia, causing an unprecedented outbreak, with 44 human cases and 7 fatalities. While a range of JEV mouse models have been reported, none of them infect adult mice with a genotype 4 isolate. The efficacy of current vaccines for this JEV genotype are also unclear. Added value of this studyWe establish well characterised adult and subcutaneously infected mouse models for JEV which recapitulate many aspects of human disease including lethal neuroinvasive infection and severe histopathological lesions. Prolonged viremia was significantly associated with lethal neuroinvasiveness in Irf7-/-mice. We demonstrate that a genotype 4 Australian isolate, JEVNSW/22, exhibited markedly diminished lethal neuroinvasion compared to other JEV genotypes. Using serum from Imojev vaccine recipients, neutralizing antibodies against JEVNSW/22 were present, albeit at sub-optimal titers. Implications of all the available evidenceThe establishment of well characterised adult mouse models of JEVNSW/22 with rare neuropenetrance after peripheral inoculation that recapitulate human disease is an important tool that can now be deployed in pre-clinical studies and to understand disease pathogenesis. Our study suggests that new vaccines should be developed against circulating JEV strains for optimal neutralizing antibody responses.

microbiology↗

bioRxiv2022

Omicron BA.5 infects human brain organoids and is neuroinvasive and lethal in K18-hACE2 mice

The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neuroinvasiveness, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. Although fulminant brain infection is not a feature of COVID-19, evidence for brain infection and brain damage in some COVID-19 patients with severe disease is becoming compelling, with the results herein suggesting that evolving omicron variants may have increasing intrinsic neuropathogenic potential.

microbiology↗

bioRxiv2020

New look at RSV infection : tissue clearing and 3D imaging of the entire mouse lung at cellular resolution.

BackgroundRespiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis mostly relied on chest x-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus-host interactions and related acute respiratory disease, at the level of the entire lung. MethodsBy combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. ResultsWhole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. ConclusionsWhole lung clearing and 3D deep-imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses.

pathology↗

bioRxiv2018

Immunological characterization of a rat model of Duchenne’s disease and demonstration of improved muscle strength after anti-CD45RC antibody treatment

Duchenne muscular dystrophy (DMD) has as standard pharmacological therapy with corticoisteroids (CS) that decrease inflammation and immune responses present in patients and animal models. CS have however limited efficacy and important and numerous side effects. Therefore, there is a need for new anti-inflammatory and pro-tolerogenic treatments that could replace or decrease doses of CS. We first assessed the status of immune system of dystrophin-deficient rats (Dmdmdx) that closely reproduce the phenotype of DMD patients. Dmdmdx rats showed increased leukocyte infiltration in skeletal and cardiac muscles, containing mostly macrophages but also T cells, and increased expression of several cytokines. Anti-CD45RC Monoclonal antibody (Mab) treatment induced immune tolerance in models of organ transplantation and GVHD (Graft Versus Host Disease). We observed that muscles and blood of DMD patients contained T CD4+ and CD8+ expressing high levels of CD45RChigh cells. Treatment of young Dmdmdx rats with anti-CD45RC MAb corrected skeletal muscle strength associated to a depletion of effectors CD45RChigh T cells with no obvious side-effects. Prednisolone treatment of Dmdmdx rats similarly increased skeletal muscle strength and was also associated to a depletion of effectors CD45RChigh cells but resulted in severe weight loss.\n\nOverall, Dmdmdx rats display important immune inflammatory response and thus represent a useful model to analyze new anti-inflammatory and tolerogenic treatments for DMD. As an example, a new treatment with anti-CD45RC antibodies improved muscle strength in Dmdmdx rats as prednisolone did but without side effects. Anti-CD45RC therapy could complement other therapies in DMD patients.

immunology↗