Biology subjects

Zillinger, T.

Publications and source records attributed to Zillinger, T..

9 recordsLinked to original sources

bioRxiv2026

Mechanical stress-induced release of mitochondrial double-stranded RNA activates RIG-I to drive osteogenic differentiation in aortic disease

Background: Gain-of-function mutations in RIG-I and MDA5 (RIG-I-like receptors, RLR) cause Singleton-Merten syndrome, an interferonopathy with early aortic calcification. Here we set out to study a possible involvement of RIG-I-like receptor signaling in the formation of aortic valve stenosis in general. Methods and results: We investigated whether mechanical stress-induced RLR activation promotes osteogenic differentiation and calcification of primary human aortic cells, whether such RLR activation drives the development of aortic valve stenosis in vivo, and whether inhibition of downstream JAK/STAT activation ameliorates aortic valve stenosis. We found that activation of primary human aortic interstitial cells by exogenous RIG-I- and MDA5 agonists induced a type I interferon response and an osteogenic program including increased expression of BMP-2 and BSP2, and promoted calcification. These effects were RIG-I/MDA5 or MAVS-dependent, as assessed by CRISPR/Cas9 knockouts. The stress-granule protein G3BP1 and the Wnt receptor FZD5 were induced downstream of RLR activation and were required for osteogenic gene expression and calcification. To assess a potential endogenous source of RIG-I activation, mechanical stretch mimicking valvular tissue stress was applied in cell cuture, which induced a significant RIG-I-dependent interferon response. Sequencing of highly purified preparations of double-stranded RNA from stretched cells demonstrated a predominant mitochondrial origin of double-stranded RNA, and depleting mitochondrial RNA abolished the stretch-dependent response. The JAK/STAT inhibitor ruxolitinib suppressed the RIG-I-induced response including the expression of osteogenic factors in vitro. In a murine wire-injury model of aortic stenosis, MAVS deficiency attenuated valvular morphological changes and improved hemodynamic parameters. Ruxolitinib attenuated hemodynamic progression, prevented aortic stenosis development, and improved survival. Conclusions: Altogether, our results demonstrate that the mechanical-stress-induced release of mitochondrial double-stranded RNA activates RIG-I signaling in aortic interstitial cells, thereby driving osteogenic differentiation and calcification via a MAVS- and G3BP1-dependent pathway. JAK/STAT inhibition with ruxolitinib mitigates this process in a mouse model of aortic valve stenosis, identifying a candidate therapeutic strategy.

cell biology↗

bioRxiv2026

The actin motor protein MYO10 facilitates post-entry spread of respiratory syncytial virus

Human respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants. However, host factors that influence disease severity remain incompletely defined. While clinical risk factors are known, identifying genetic susceptibility has been challenging. In this study, we combined human genetics with functional virology to identify host factors that modulate RSV infection and spread. Starting from a cohort of infants hospitalized with severe RSV disease, we prioritized rare coding variants present in homozygous form and predicted to cause strong functional impairment, and selected candidate genes for mechanistic follow-up. Functional interrogation of 23 candidates by CRISPR/Cas9 knockout screening in human lung epithelial cells identified unconventional myosin-X (MYO10), encoding the actin-based motor protein myosin-X, as a critical host factor for RSV. Genetic disruption or siRNA-mediated depletion of MYO10 significantly reduced RSV infectivity, with the strongest effects at post-entry stages of the viral life cycle. Loss of MYO10 impaired filopodia formation, cell migration, and wound healing, leading to altered cell-cell connectivity and restricted viral dissemination. MYO10 depletion reduced both short-range cell-to-cell transmission and longer-distance extracellular spread, resulting in fewer infected cells and diminished accumulation of progeny virus in culture supernatants. In contrast, RSV entry, early gene expression, and interferon responses were unaffected. Finally, a rare homozygous MYO10 motor-domain variant (rs7737765; H148Y), enriched in severe cases, also reduced RSV replication in cell culture--opposite to expectations for a risk allele--yet underscoring biological relevance and suggesting that MYO10 variation may influence disease in vivo through additional effects on epithelial function. ImportanceRespiratory syncytial virus (RSV) is a major cause of severe respiratory illness in infants, yet it remains unclear why some children develop more serious disease than others. In this study, we combined patient genetic data with laboratory experiments to identify host factors that influence how RSV spreads in the lung. We found that the human protein MYO10, which mediates the formation of small cell protrusions, plays a key role in enabling the virus to spread from cell to cell. When MYO10 was disrupted, viral spread was strongly reduced, even though early steps of infection were unaffected. Interestingly, a rare genetic variant in MYO10 found in patients also altered viral replication, highlighting potential clinical relevance. These findings provide new insight into how host cell architecture contributes to RSV infection and suggest that targeting host pathways involved in viral spread could complement existing antiviral strategies.

microbiology↗

bioRxiv2026

Inositol hexakisphosphate Functions as a Cofactor and Modulator of ADAR1 Activity

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn{superscript 2}-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

molecular biology↗

bioRxiv2022

RIG-I activation primes and trains innate antiviral immune memory

Adaptive processes of the innate immune system, known as trained immunity (TI), are critical to human health and disease, yet they have not been systematically investigated downstream of antiviral sensing. Here, we elucidate the potential of the antiviral cytosolic RNA receptor retinoic acid-inducible gene I (RIG-I) to train, prime and tolerize the innate immune system. Using a specific RIG-I agonist, we observed that repetitive stimulation enhanced interferon-stimulated gene (ISG) and pro-inflammatory cytokine induction in human primary monocytes, epithelial cells and fibroblasts and afforded non-specific antiviral protection. RNA sequencing revealed broad, cell type-specific transcriptional changes, indicative of priming of ISGs and training of the NF{kappa}B pathway, without measurable tolerization, while ATAC sequencing in monocytes demonstrated chromatin remodeling and enhanced accessibility of key transcription factor-binding motifs such as STAT1. Moreover, while STAT1 signaling was critically required, it was not sufficient to recapitulate RIG-I induced TI. Altogether, our data demonstrate that RIG-I-mediated TI promotes an immunologically alert state with important implications for host defense and the application of RIG-I ligands in anti-infective and anti-tumoral therapies. One Sentence SummaryRIG-I activation trains and primes innate immune response at the cellular level, affording non-specific immune protection by immune and non-immune cells.

immunology↗

bioRxiv2022

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5triphosphorylated and 5base-paired RNA(dsRNA). Here, we found that, while 5unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5-monophosphate(5p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with a long, highly structured, polyA-bearing, non-coding mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5p-RNA recognition is crucial to preventing mtRNA-triggered RIG-I-mediated autoinflammation.

immunology↗

bioRxiv2022

SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutieres syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here we show that SAM domain and HD domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

immunology↗

bioRxiv2022

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased activity of the DNA sensor cGAS and enhanced IFN-I signature. To identify potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-{beta}, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-{beta}-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcome. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/493983v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@521964org.highwire.dtl.DTLVardef@13506org.highwire.dtl.DTLVardef@1403dbeorg.highwire.dtl.DTLVardef@958cc4_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryKessler et al. identify aberrant DNA recognition by cGAS/STING axis and IFN-I production by inflammatory macrophages as a major driver of severe ANCA-associated vasculitis (AAV). Pharmacological interventions blocking this pathway ameliorate disease and accelerate recovery, identifying potential targets for therapeutic intervention in patients.

immunology↗

bioRxiv2022

cGAS/STING-DEPENDENT SENSING OF ENDOGENOUS RNA

Defects in nucleic acid metabolizing enzymes lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing a pathogenic type I interferon response and inflammatory diseases. In these pathophysiological conditions, cGAS-driven IFN production is linked to spontaneous DNA damage. Physiological, or tonic, IFN signaling on the other hand is essential to functionally prime nucleic acid sensing pathways. Here we show that low-level chronic DNA damage in mice lacking the Aicardi-Goutieres syndrome gene SAMHD1 reduced tumor-free survival when crossed to a p53-deficient, but not to DNA mismatch repair-deficient background. Increased DNA damage did not result in higher levels of type I interferon. Instead, we found that the chronic interferon response in SAMHD1-deficient mice was driven by the MDA5/MAVS pathway but required functional priming through the cGAS/STING pathway. Our work positions cGAS/STING upstream of tonic IFN signaling and highlights an important role of the pathway in physiological and pathophysiological innate immune priming. SummaryLoss of the dNTPase and DNA repair enzyme SAMHD1 is associated with cancer and causes systemic autoimmunity. We show transformation-promoting spontaneous DNA damage and MDA5-driven but cGAS/STING-dependent chronic type I interferon production in SAMHD1-deficient mice.

immunology↗

bioRxiv2020

Human IFITM3 restricts Chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction

Interferon-induced transmembrane (IFITM) proteins restrict infection by enveloped viruses through interfering with membrane fusion and virion internalisation. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies remain largely unexplored. Here, we characterized the impact of IFITM proteins and variants on entry and spread of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in human cells, and provide first evidence for a CHIKV-mediated antagonism of IFITM proteins. IFITM1, 2 and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and during cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in the loss of its antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that has been proposed to associate with severe influenza in humans, restricted CHIKV, MAYV and influenza A virus infection as efficiently as wild-type IFITM3. Finally, all antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several non-structural protein(s) of CHIKV.

microbiology↗