Biology subjects

Zacchigna, S.

Publications and source records attributed to Zacchigna, S..

5 recordsLinked to original sources

bioRxiv2026

Targeting IL-2 to inflamed tissues via oxidation-specific epitopes enables third-generation bispecific IL-2 therapeutics

Interleukin-2 (IL-2) is essential for the survival and activation of regulatory T cells (Tregs). Low-dose native IL-2 (IL-2LD) therapy restores immune regulation in vivo and has shown reproducible clinical benefit across multiple autoimmune, inflammatory, and neuroimmune diseases. Attempts to improve IL-2 through engineered variants (muteins) have mainly focused on enhancing Treg selectivity by reducing IL-2 receptor {beta}-chain binding, but this strategy profoundly diminishes biological potency, likely contributing to the limited clinical efficacy of IL-2 muteins. Here, we develop a ''third-generation IL-2'' that combines site-specific targeting and bifunctionality. We generated a bivalent fusion protein linking IL-2 to a single-chain antibody recognizing oxidation-specific epitopes (OSEs), which are abundantly expressed at inflamed sites. Targeting OSEs provides not only site-specific localization, but also true bifunctionality as both anti-OSE antibodies and IL-2LD independently show therapeutic benefit in limiting inflammation. We first show that IL-2IT has bifunctional biological activities in vitro. In vivo, IL-2IT had increased specificity for Treg over Teff activation, which we attribute to a conformation-dependent modulation of IL-2 receptor engagement. Importantly, IL-2IT provided precise delivery to inflamed tissues in models of psoriasis and colitis. Altogether, this resulted in superior therapeutic benefit in multiple clinical settings, including in atherosclerosis models. Thus, our strategy illustrates a generalizable approach to cytokine engineering that preserves native signaling while achieving spatial control. Specifically, our findings validate OSE targeting as an efficient strategy to guide therapeutics to sites of inflammation and establish OSE-IL-2 as a promising bispecific Treg engager for treating inflammation.

immunology↗

bioRxiv2025

Adventitial leptin receptor-expressing fibroblasts are preferential contributors to fibrotic remodeling of the heart post infarction

BackgroundCardiac fibrosis, a hallmark of heart failure and an unmet clinical need, arises from pathological activation of pre-existing cardiac fibroblasts (CFs), but the contribution of CF heterogeneity to this process remains unclear. MethodsMurine models were used to lineage trace or deplete a specific sub-population of CFs at baseline and after myocardial infarction (MI). Transcriptional and epigenetic differences between fibroblast subsets were assessed using next-generation sequencing. Conservation in humans was evaluated through single-cell RNA-seq datasets and histological examination. ResultsIn mice, fibroblasts were the sole cardiac cell type expressing the signaling-capable isoform of the leptin receptor (LepR). LepR+ CFs emerged neonatally, occupied a defined niche in the coronary adventitia, exhibited enhanced hedgehog signaling, and responded to leptin. After MI, LepR-Cre+ CFs proliferated more than interstitial CFs, became a predominant fibroblast lineage in the scar, and their genetic ablation reduced fibrosis while improving function. LepR+ CFs were also detected in the human heart, where they were embedded in an adipocyte-rich niche. ConclusionsThese findings identify adventitial fibroblasts as key drivers of pathological remodeling and demonstrate that fibroblasts, rather than cardiomyocytes, are the principal responders to leptin in the heart, redefining how this major endocrine pathway influences cardiac remodeling and disease.

cell biology↗

bioRxiv2023

Tissue-resident uterine regulatory T cells support fetal growth

Regulatory T cells (Tregs) are known to contribute to successful allogeneic pregnancy by promoting maternal-fetal tolerance. Here, phenotypic studies and parabionts identified a population of uterine tissue-resident Tregs (uTregs) with trophic functions in the non-pregnant mouse endometrium. Phenotypically, uTregs are CD25-/lo and they present characteristic phenotypic attributes of tissue-resident memory T cells with effector functions. Transcriptionally, uTregs are distinguishable from other peripheral or tissue-resident Tregs by their expression of genes related to extracellular matrix remodeling and vasculogenesis, and they express a polyclonal T cell receptor (TCR) repertoire. Pregnancy triggers the expansion of uTregs, which remain CD25-/lo, retain their polyclonal TCR repertoire, and increase the expression of genes involved in regulation of hypoxia and vasculogenesis. Functionally, we show that uTregs support the proliferation of primary uterine microvascular endothelial cells in vitro and that a specific depletion of uTregs in pregnant mice leads to fetal intra-uterine growth retardation (IUGR). Thus, a previously unidentified population of CD25-/lo uTregs promotes uterine tissue remodeling before and during pregnancy, and contributes to fetal growth.

immunology↗

bioRxiv2021

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet pro-coagulant activity

BackgroundThrombosis of the lung micro-vasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting this activity. As TMEM16F plays an important role in the stimulation of the pro-coagulant activity of platelets, and considering that platelet abnormalities are common in COVID-19 patients, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. MethodsWe produced SARS-CoV-2 Spike or VSV-G protein-pseudotyped virions, or generated cells expressing Spike on their plasma membrane, and tested their effects on platelet adhesion (fluorescence), aggregation (absorbance), exposure of phosphatidylserine (flow cytometry for annexin V binding), calcium flux (flow cytometry for fluo-4 AM), and clot formation and retraction. These experiments were also conducted in the presence of the TMEM16F activity inhibitors Niclosamide and Clofazimine. ResultsHere we show that exposure to SARS-CoV-2 Spike promotes platelet activation, adhesion and spreading, both when present on the envelope of virions or upon expression on the plasma membrane of cells. Spike was effective both as a sole agonist or by enhancing the effect of known platelet activators, such as collagen and collagen-related peptide. In particular, Spike exerted a noticeable effect on the procoagulant phenotype of platelets, by enhancing calcium flux, phosphatidylserine externalisation, and thrombin generation. Eventually, this resulted in a striking increase in thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine almost abolished this Spike-induced pro-coagulant response. ConclusionsTogether, these findings provide a pathogenic mechanism to explain thrombosis associated to COVID-19 lung disease, by which Spike present in SARS-CoV-2 virions or exposed on the surface of infected cells, leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing drugs targeting this protein, such as Niclosamide, for COVID-19 therapy.

pathology↗

bioRxiv2021

A polyphenol-rich extract of Olive Mill Wastewater Enhances cancer chemotherapy effects while mitigating cardiac toxicity.

BackgroundCardiovascular toxicities still remain one of the most undesirable side effects in cancer patients receiving chemotherapy, and cardiotoxicity has been detected associated with many therapeutic regimens. A number of mechanisms are reported for these effects, some of which are related to inflammation, oxygen radical generation, mitochondrial damage. Extra-virgin olive oil (EVOO) is rich in cancer preventive polyphenols endowed with anti-inflammatory, antioxidant activities which could exert protective effects on the heart cells. One very interesting derivative of EVOO preparation is represented by purified extract form waste waters. Here, we investigated the anti-cancer activity when combined with chemotherapeutics as well as potential cardioprotective activities of a polyphenol-rich extract from waste product of the EVOO, named A009. Methods and ResultsMice bearing prostate cancer (PCa) xenografts were treated with cisplatin with and without A009. Tumor cell growth was reduced by cis and by A009 and further hindered by the combination. The effects of the A009 extract on cardiovascular toxicities was investigated in vivo. Hearts of mice were analyzed, and the mitochondria were studied by transmission electron microscopy. A protection activity by A009 was observed. To confirm the in vivo data obtained with cisplatin therapy, tumor cell lines and rat cardiomyocytes were treated with cisplatin in vitro with and without A009. A009 enhanced cisplatin and 5FU reduced cancer cell growth while did not further affect co-treated rat cardiomyocytes. Another frequently used chemotherapeutic agent 5-fluorouracil (5FU), was also tested in this assay a similar effects were observed. The cardioprotective effects of the A009 extract towards 5 FU chemotherapy were further investigated in a second system of in vitro cultures, on cardiomyocytes freshly isolated from mice pups. These cells were treated with 5-fluorouracil and A009. Wastewater extract mitigated toxicity of the fluorpyrimidine. ConclusionsIn vivo, we found synergisms of A009 and cisplatin in prostate cancer treatment. Hearts of mice xenografted with PCa cell lines and receiving co-treatments of A009 extracts along with cisplatin had reduced mitochondria damage compared to chemotherapy alone, indicating a cardioprotective role. A009 in vitro was additive to cisplatin and 5FU to reduce cancer cell growth while did not further affect rat cardiomyocytes cell cultures treated with cisplatin and 5FU. The A009 extract also rescued the proliferation rate of neonatal murine cardiomyocytes treated with 5-Fluorouracil. Our study demonstrates that the polyphenol rich purified A009 extracts enhances the effect of chemotherapy in vitro and in vivo but mitigates effects on heart and heart cells. It could therefore represent a potential candidate for cardiovascular prevention in patients undergoing cancer chemotherapy.

cancer biology↗