Biology subjects

Del Galdo, F.

Publications and source records attributed to Del Galdo, F..

11 recordsLinked to original sources

bioRxiv2026

Chikungunya virus live-cell reporter systems reveal cell-type-dependent antiviral responses to ribavirin

Chikungunya virus (CHIKV) remains a significant global public health threat, yet antiviral discovery is limited by experimental systems that provide little mechanistic resolution and rely heavily on transformed cell lines of uncertain physiological relevance. Here, we developed complementary live-cell CHIKV reporter systems comprising a dual-fluorescent subgenomic replicon and fluorescent infectious viruses that enable real-time monitoring of distinct stages of the viral replication cycle. By coupling an nsP3-tagged reporter to genomic RNA translation and replication with a subgenomic reporter of structural gene expression, we achieved temporal resolution of early and late replication events in living cells. Functional validation using replication-defective and subgenomic RNA-deficient mutants confirmed that the system distinguishes between viral RNA replication and downstream structural gene expression. Application of these reporters revealed distinct antiviral responses across transformed and physiologically relevant human cell models. Ribavirin and the nsP2 inhibitor RA-0002034 potently inhibited CHIKV replication in transformed cell lines. Unexpectedly, ribavirin activity was markedly diminished in primary human skeletal muscle myoblasts and dermal fibroblasts, despite robust antiviral effects in commonly used screening cell lines. Mechanistic studies showed that ribavirin-mediated inhibition was rescued by guanosine but not inosine supplementation and was recapitulated by mycophenolic acid, consistent with guanine nucleotide depletion as the predominant antiviral mechanism under the conditions tested. Notably, inhibition through this pathway was substantially less effective in primary human cells. Together, these findings establish versatile live-cell CHIKV reporter systems for mechanistic studies of viral replication and reveal striking cell-type-dependent differences in response to guanine nucleotide depletion. Our results demonstrate that host-cell context can profoundly influence both CHIKV replication and antiviral susceptibility, highlighting the importance of physiologically relevant models for understanding virus-host interactions and antiviral mechanism.

microbiology↗

bioRxiv2026

Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

BackgroundSystemic sclerosis (SSc) is a severe autoimmune disease characterised by progressive fibrosis driven by fibroblast activation. Primary cilia, key hubs for profibrotic signalling, are markedly shortened in SSc fibroblasts, but the mechanisms underlying this phenotype remain unclear. This study aimed to define the signalling pathways responsible for primary cilia shortening and fibroblast activation in SSc. MethodsPrimary dermal fibroblasts from SSc patients and healthy controls were analysed for cilia incidence and length by immunofluorescence, profibrotic marker expression by qPCR, and contractility using gel contraction assays. Cells were treated with TGF{beta}1 and pharmacological inhibitors targeting AURKA, HDAC6, ROCK2, and Smad3 signalling. CAV1-silenced fibroblasts were used as an in vitro model of SSc. ResultsMaintenance of the constitutively short primary cilia phenotype in SSc fibroblasts did not require active TGF{beta} signalling. However, TGF{beta}1 induced reversible cilia shortening in healthy fibroblasts and further shortened cilia in SSc fibroblasts to a similar final length, mediated by Rho/ROCK2 rather than canonical Smad3-dependent signalling. Constitutive cilia shortening in SSc was driven by aberrant AURKA activity upstream of HDAC6, promoting ciliary disassembly. Pharmacological inhibition of AURKA or HDAC6 selectively elongated cilia in SSc fibroblasts, reduced profibrotic marker expression, and abrogated fibroblast contractility. CAV1-silenced fibroblasts similarly exhibited constitutive cilia shortening that was reversed by AURKA inhibition without affecting healthy cells. ConclusionsAberrant activation of the AURKA/HDAC6 axis maintains short primary cilia and promotes fibroblast activation in SSc. These findings reveal a mechanistic link between cilia morphology and fibrosis and identify AURKA as a potential therapeutic target for SSc-associated tissue remodelling.

cell biology↗

bioRxiv2026

Imbalance of Ciliary Programs Drives Fibroblast Differentiation and Fibrotic Signaling in Systemic Sclerosis

Systemic sclerosis (SSc) is a chronic fibrotic disease characterized by accumulation of activated profibrotic myofibroblasts in multiple organs. The mechanisms triggering pathogenic fibroblast to myofibroblast reprogramming in SSc, and maintaining the activated myofibroblast state, are not well known. Recent studies show that primary cilia (PC), solitary sensory organelles that integrate diverse chemical and mechanical signaling pathways, may regulate fibroblast fates. Here, using an orthogonal multimodal strategy spanning human cohorts, single-cell trajectories, and targeted perturbation models, we identify a fundamental imbalance in cilia-program dynamics as a unifying driver of fibrotic activation in SSc. Meta-analysis of skin biopsies integrating eight microarray datasets and two independent scRNA-seq cohorts, revealed a conserved 15-gene cilia signature that is altered in SSc. Single-cell trajectory mapping resolved a principal progenitor[->] secretory-like [->] myofibroblast differentiation axis, where SSc fibroblasts displayed spatially dysregulated ciliary programs that established a prolonged, disassembly-dominant "cilia-off" state. Notably, this ciliary imbalance appeared to emerge before the transition to fibrotic gene programs, positioning cilia disruption as an initiating, not secondary, event in fibroblast programming. Consistent with these transcriptomic findings, PC length was significantly reduced in SSc skin and cultured fibroblasts. Mechanistically, genetic and pharmacological perturbation studies demonstrated that cilia disruption is sufficient to drive fibrotic programs. These data establish a reciprocal regulatory framework in which shortened cilia promote sustained activation of TGF-{beta}-Hippo feed-forward signaling, driving fibroblast-to-myofibroblast transition and amplifying fibrotic responses. Together, these findings position the imbalance between ciliary assembly and disassembly as a central determinant of fibrotic fibroblast fate in SSc. Moreover, they indicate that therapeutically stabilizing primary cilia, "ciliotherapy", can reset pathological fibroblast trajectories and represents a promising antifibrotic strategy for SSc and other fibrotic conditions. TeaserDisrupted Primary Cilia Drives Pathophysiology of Systemic Sclerosis.

cell biology↗

bioRxiv2025

Targeting Tenascin-C-Toll-like Receptor 4 signalling with Adhiron-derived small molecules - a viable strategy for reducing fibrosis in Systemic Sclerosis

Tissue fibrosis is a hallmark of systemic sclerosis (SSc) and results from the excessive production and deposition of collagen and other extracellular matrix proteins by resident fibroblasts. This excessive connective tissue accumulation leads to tissue disruption and subsequent dysfunction in the skin, lungs and other internal organs. Recent studies highlight a role for the matricellular protein Tenascin C in SSc, whereby its stimulation of Toll-like receptor 4 triggers self-sustaining fibroblast activation and ensuing fibrosis. We have utilised Adhiron guided ligand discovery to generate small molecules that target the fibrinogen-like globe domain of Tenascin C, a region involved in Toll-like receptor 4 activation and have demonstrated a reduction in the profibrotic phenotype of human dermal fibroblasts. These studies may facilitate the development of effective targeted therapy for fibrosis in SSc and other fibrotic diseases. Moreover, it highlights the utility of Adhiron guided ligand discovery to generate small molecule inhibitors to selectively modulate proteins. HighlightsO_LIAdhiron screening was undertaken to identify binders to the FBG domain of Tenascin C and the crystal structure of the FBG-Adhiron binding was resolved. C_LIO_LIAdhiron guided ligand discovery was used to generate small molecule inhibitors that selectively target the FBG domain of Tenascin C. C_LIO_LISPR was used to show binding of these molecules to the FBG domain of Tenascin C C_LIO_LIThe small molecule inhibitors generated attenuated TGF{beta}-mediated fibrosis in dermal fibroblasts C_LI

physiology↗

bioRxiv2024

Molecular glues that inhibit specific Zn2+-dependent DUB activity and inflammation

Deubiquitylases (DUBs) are crucial in cell signalling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signalling by cleaving K63-linked polyubiquitin chains on Type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN DUB, BRCC36 is challenging to target with selective inhibitors. We discovered first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilise a 16-subunit BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit SHMT2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild type BRISC, and this effect was absent when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential new strategy to mitigate Type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting, rather than blocking, protein-protein interactions.

biochemistry↗

bioRxiv2024

Chloride intracellular channel 4 (CLIC4) is a global regulator of type 1 interferon signaling in Systemic Sclerosis (SSc) epithelial cells

ObjectivesSystemic sclerosis (SSc) is an autoimmune disease in which an immune-related injury induces fibrosis of the skin, progressing to affect the internal organs in the most serve cases. Type 1 interferon (IFN) signaling plays a major role in SSc disease progression. We have previously shown the chloride intracellular channel 4 (CLIC4) is upregulated in SSc skin fibroblasts and plays an important role in SSc fibrosis. In this study we investigated the role of CLIC4 in SSc keratinocyte biology. Methodshealthy (HC) and SSc skin biopsies were analysed by immunohistochemistry for the expression of CLIC4. The skin keratinocyte cell line Hacats was stimulated with a range of type 1 IFN signaling agonists (POLY I:C, POLY dA:Dt, ODN 2216 and IFN-). CLIC4 was inhibited with the chloride channel inhibitors NPPB and IAA-94 or siRNA. Conditioned media from HC or SSc fibroblasts was employed for indirect co-culture of Hacats and HUVECs. ResultsSSc skin biopsies showed high levels of CLIC4 in SSc skin fibroblasts, keratinocytes and endothelial cells compared to HC. Co-culture of Hacats and Huvecs with SSc fibroblast media induced CLIC4 expression. CLIC4 played an important role in type 1 IFN signalling in keratinocytes. Inhibition of CLIC4 blocked TLR3, TLR9 and cGAS mediated activation of the type 1 IFN signaling pathway. Additionally, inhibition of CLIC4 prevented SSc fibroblast media from inducing a type 1 IFN response in keratinocytes. ConclusionsThe data presented in this study suggests CLIC4 is a global regulator of type 1 IFN signalling in SSc epithelial cells. Key MessagesO_ST_ABSWhat is already knownC_ST_ABSSSc disease progression is driven in part by a Type 1 IFN signature and CLIC4 has previously been implicated in SSc fibroblast activation. What this study addsWe show for the first time CLIC4 is a regulator of type 1 interferon signalling in epithelial cells and plays an important role in the signalling found in SSc skin. How this study might affect researchTargeting CLIC4 in the context of SSc may disrupt the fibrosis and inflammation associated with SSc.

immunology↗

bioRxiv2024

Morphological Reprogramming of Primary Cilia Length Mitigates the Fibrotic Phenotype in fibroblasts across diverse fibrotic conditions

Fibrosis is a hallmark of systemic sclerosis (SSc) and many diverse and incurable diseases. Myofibroblast activation, a common cellular phenomenon shared across fibrotic diseases, is marked by actin polymerization known to affect primary cilia (PC) length. We discovered that fibroblasts from diverse fibrotic conditions display significantly reduced PC length ex vivo. Treatment of healthy fibroblasts with profibrotic TGF-{beta}1 induced PC shortening, while silencing ACTA2 in SSc skin fibroblasts caused PC elongation. Importantly, we found that PC length is negatively correlated with cellular expression of -SMA in TGF-{beta}1-stimulated healthy fibroblasts, or pharmacologically de-differentiated myofibroblasts. PC elongation by microtubule polymerization induction in SSc skin fibroblasts using LiCl or the HDAC6 inhibitor tubacin, reversed and mitigated fibrotic responses. Our results implicate a key role for microtubule polymerization in restraining fibrotic responses and suggest that modulation of PC dynamics may represent a potential therapeutic strategy for SSc and other treatment-resistant diseases associated with fibrosis. Teaser. PC length shortening is a hallmark of fibrosis.

cell biology↗

bioRxiv2023

Systemic Sclerosis dermal fibroblast exosomes trigger a Type 1 interferon response in keratinocytes through the TBK/JAK/STAT signalling axis

BackgroundActivation of Type I IFN response has been shown to correlates with disease activity in systemic sclerosis. It is currently unknown whether the tissue-specific Type I IFN activation is a consequence of the response observed in blood or rather its source. Exosomes from SSc fibroblasts were recently shown to activate macrophages in vitro. Here, we aimed to determine the source of Type I IFN signature in SSc skin biopsies and the potential role of exosomes from SSc dermal fibroblasts in the process. MethodsSkin biopsies were obtained from healthy and SSc patients forearms and processed for dermal fibroblasts and keratinocytes. Exosomes were isolated from healthy and SSc dermal fibroblast supernatants by ultracentrifugation and added to human skin keratinocytes. Keratinocyte transcriptome was analysed by RNA-seq analysis. TANK-binding kinase (TBK) and JAK were inhibited using a small molecule inhibitor (GSK8612) and Tofacitinib, respectively. ResultsSSc skin biopsies showed highest levels of Type I IFN response in the epidermal layer. RNA-seq analysis of keratinocytes transcriptome following exposure to dermal fibroblast exosomes showed strong upregulation of IFN signature genes induced by SSc exosomes compared to Healthy control. Inhibition of TBK or JAK activity suppressed the upregulation of the IFN signature induced by SSc exosomes. ConclusionIFN activation of SSc keratinocytes is dependent on their crosstalk with dermal fibroblasts and inducible by extracellular exosomes. Our data indicates that SSc fibroblasts exosomes may carry the signal zero of local Type I IFN activation through activation of pattern recognition receptors upstream of TBK. Key MessagesO_LISSc patient skin exhibit a type 1 IFN signature with keratinocytes being the major source of the signature C_LIO_LICross talk between the fibroblasts and keratinocytes through exosomes may be signal zero for the type 1 IFN signature C_LIO_LIBlocking JAK in the keratinocytes with Tofacitinib disrupts the type 1 IFN signature C_LI

immunology↗

bioRxiv2022

The Beta secretase BACE1 drives Systemic Sclerosis fibroblasts activation through B-catenin and Notch signalling

The beta-amyloid precursor protein cleaving enzyme 1 (BACE1) is well known for its role in the development of Alzheimers disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in SSc patient skin. Inhibition of BACE1 with small molecule inhibitors or siRNA blocked SSc and fibrotic stimuli mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on {beta}-catenin and Notch signalling. The Neurotropic factor BDNF negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from SSc patients show higher A{beta} and lower BDNF levels compared to healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a new therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimers disease and could be repurposed to ameliorate fibrosis progression.

immunology↗

bioRxiv2021

Chloride intracellular channel 4 (CLIC4) expression is transcriptionally regulated by crosstalk of the TGF-beta, Wnt and Hedgehog signalling pathways

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts. CLIC4 expression and activity are regulated by TGF-{beta} signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show Wnt3a/{beta}-catenin and Smoothened/GLI signalling cooperate with SMAD3 to regulate CLIC4 expression in normal dermal fibroblasts, and that inhibition of SMAD3 expression or activity abolishes Wnt and Hh-dependent CLIC4 induction. We further show that expression of the profibrotic marker -smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Our data highlight novel mechanisms that regulate CLIC4 expression that present targetable pathways to prevent fibroblast activation in SSc and other fibrotic conditions.

cell biology↗

bioRxiv2020

Targeting BDCA2 suppresses Plasmacytoid dendritic cell dependent interferon response and tissue fibrosis

Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of Scleroderma (SSc) through their ability to infiltrate the skin and secrete interferons (IFN) and proinflammatory chemokines. Blood Dendritic Cells Antigen 2 (BDCA2) is an inhibitory type II C-type lectin expressed by human pDC. Here we determined the effects of BDCA2 internalisation on pDC mediated skin inflammation and fibrosis in human preclinical models of skin inflammation and fibrosis in vitro and in vivo. BDCA2 targeting reversed TLR-signalling induced transcriptome and differentiation of pDC and suppressed their ability to induce IFN response in organotypic 3D human skin cultures in vitro. In vivo, xenotransplantation of human pDC into immunocompromised mice (XenoSCID) significantly increased IFN induced responses to topical TLR7/9 agonist and separately enhanced the fibrotic response to bleomycin. Targeting of BDCA2 strongly suppressed both of these pathological responses ameliorating skin inflammation and fibrosis. Together, these preclinical data strongly support the notion that human pDC play a key role in immune driven skin fibrosis, which can be effectively blocked by targeting BDCA2.

immunology↗