Biology subjects

Zanoni, I.

Publications and source records attributed to Zanoni, I..

16 recordsLinked to original sources

bioRxiv2025

Chronic sensing of host-derived lipids is an all-in-one signal that primes and activates NLRP3.

Activation of the NLRP3 inflammasome leads to the production of bioactive interleukin (IL)-1{beta} fostering atherosclerosis. The current dogma is that NLRP3 must be first primed by microbial stimuli, known as pathogen-associated molecular patterns (PAMPs), and then activated by either microbial or host-derived inflammatory cues. The mechanism that controls NLRP3 functioning in the context of non-communicable diseases lacking overt microbial infections remains debated. Here, we show that chronic exposure to atherosclerosis-associated oxidized phospholipids (oxPLs) simultaneously primes and activates NLRP3 independently of microbial cues. Mechanistically, chronic exposure to host-derived oxPLs activate the transcription factor NRF2, which is necessary and sufficient to prime and activate NLRP3 in a PAMP-independent manner. NRF2 chronic activation drives oxidized mitochondrial DNA to activate NLRP3. Ex vivo analyses of atherosclerotic plaques in mice and humans identify a population of monocytes-derived macrophages which activates NRF2 and expresses IL-1{beta}. Overall, our data point to oxPL-dependent NRF2 activation as an all-in-one signal necessary and sufficient to prime and activate NLRP3, sustaining atherogenesis.

immunology↗

bioRxiv2025

DNASE1L3 surveils mitochondrial DNA on the surface of distinct mammalian cells

The extracellular space is a critical environment for discriminating self versus non-self nucleic acids and initiating the appropriate immune responses through signaling cascades to relay information about extracellular nucleic acids. Here, we provide evidence that oxidized mitochondrial DNA is tethered to the surface of select mammalian cells through cell surface proteins and heparan sulfate proteoglycans. We demonstrate that cell surface DNA accumulates in large clusters that partially overlap with domains enriched in RNA binding proteins. Finally, we show that human and murine B cell surfaces contain DNA that can be cleared by the secreted nuclease DNASE1L3, and that patients with a DNASE1L3 missense variant associated with increased risk for autoimmune disease harbor increased levels of surface DNA on B and T cells. Taken together, this work expands the scope of cell surface nucleic acid biology and provides a mechanistic link between cell surface molecules and DNA targeting in autoimmune disease.

cell biology↗

bioRxiv2025

Group 2 Innate lymphoid cells promote allograft survival by constraining and inducing anergy in alloreactive CD4+ T cells

Although solid organ transplant outcomes have dramatically improved over the last several decades, incomplete understanding of the immune interface between the donor organ and the recipients immune system has impaired our ability to induce immune tolerance in most transplant recipients. Since group 2 innate lymphoid cells (ILC2s) reside in all transplanted solid organs, participate in wound healing, and coordinate other immunoregulatory cell populations, we investigated their role in the alloimmune response. Using a mouse heterotopic cardiac transplant model, we show that recipient ILC2s replace donors ILC2s, upregulate MHCII without expressing costimulatory molecules. In addition, recipient derived ILC2s process and present alloantigen, inducing CD4+ T cell anergy via Caspase-3 pathway. When recipient-derived ILC2s are not present, we observed a significant increase in infiltrating donor reactive CD4+ T cells and worsened allograft survival. Additionally, expansion of ILC2s in vivo through IL33 administration prolonged the survival of murine heart allografts. Overall, these data highlight a critical and novel immunoregulatory role of host-derived ILC2s in solid organ transplant, where they induce anergy in alloreactive CD4+ T cells, promoting the induction of alloimmune tolerance.

immunology↗

bioRxiv2025

Adjuvant conditioning enhances neutrophil function while inducing a suppressive peritoneal macrophage phenotype

Adjuvants are widely used to boost the immune response during vaccination protocols. Our group has previously reported that repeated intraperitoneal administration of alum in mice, known as adjuvant conditioning (AC), creates an immunosuppressive environment that delays allogeneic graft rejection through NLRP3-dependent MDSC expansion. However, little is known about the effects of AC on the reprogramming of peritoneal cavity cells, particularly the different peritoneal macrophage populations and the effects in the adaptive immune response. We found a population-specific immune response to alum, with small peritoneal macrophages (SPMs) being more prone to inflammasome activation than large peritoneal macrophages (LPMs) in vitro. In vivo, alum exposure led to NLRP3-dependent macrophage disappearance reaction (MDR) of LPMs, which could be explained by aggregate formation and migration to the omentum. AC also induced the reprogramming of resident macrophages and infiltrating monocytes towards a less inflammatory state, making them more vulnerable to bacterial infections, but recruited neutrophils with enhanced killing ability. This suggests that AC may influence both innate and adaptive immunity in distinct ways, reprogramming cells to different profiles, indicating its potential as an immunosuppressive treatment for autoimmune diseases and transplant rejection.

immunology↗

bioRxiv2024

Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

Trained immunity enhances responsiveness of the innate immune system upon restimulation. Although adjuvants are used to enhance immune responses, we showed that repeated administration of alum, termed adjuvant conditioning (AC), establishes an immunosuppressive environment that delays allogeneic graft rejection by expanding myeloid-derived suppressor cells (MDSCs). Here, we show that AC-induced MDSCs suppress antigen specific adaptive responses both in vitro and in vivo, and that the immunosuppression is abolished in the absence of NLRP3 and IL-1 signaling. Allogeneic pancreatic islets transplanted into AC-treated NLRP3-/- mice are not protected, demonstrating that AC requires NLRP3 signaling. Finally, AC also has an immunosuppressive effect on human cells. Overall, our data show that AC establishes an immunosuppressive milieu via the NLRP3/IL-1 axis, leading to trained immunosuppression, or trained tolerance. Our findings give a potent mandate to explore the possibility to target the NLRP3/IL-1 pathway as a new promising strategy to condition transplant recipients and promote allograft tolerance.

immunology↗

bioRxiv2023

Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter.

Macrophages detect invading microorganisms via pattern recognition receptors that recognize pathogen-associated molecular patterns, or via sensing the activity of virulence factors that initiates effector-triggered immunity (ETI). Tissue damage that follows pathogen encounter leads to the release of host-derived factors that participate to inflammation. How these self-derived molecules are sensed by macrophages and their impact on immunity remain poorly understood. Here we demonstrate that, in mice and humans, host-derived oxidized phospholipids (oxPLs) are formed upon microbial encounter. oxPL blockade restricts inflammation and prevents the death of the host, without affecting pathogen burden. Mechanistically, oxPLs bind and inhibit AKT, a master regulator of immunity and metabolism. AKT inhibition potentiates the methionine cycle, and epigenetically dampens Il10, a pluripotent anti-inflammatory cytokine. Overall, we found that host-derived inflammatory cues act as "self" virulence factors that initiate ETI and that their activity can be targeted to protect the host against excessive inflammation upon microbial encounter.

immunology↗

bioRxiv2022

Correction of age-associated defects in dendritic cell functions enables CD4+ T cells to eradicate tumors in the elderly

Defective host defenses later in life are associated with changes in immune system activity. The means to correct immune defects to ensure immunity in the elderly are undefined. In this study, we found that CD8+ T cells, which are necessary for anti-tumor immunity in young mice, are not required to eradicate the same cancers later in life. Rather, CD4+ T cells drive anti-tumor immunity in elderly mice. The generation of anti-tumor CD4+ T cells requires multiple dendritic cell (DC) activities that are elicited by immune agonists known as hyperactivators, but not by adjuvants that model those used clinically. DC hyperactivators correct age-associated defects in DC migration and T cell co-stimulation while enabling NLRP3 inflammasome activities within living cells. These combined activities enable DCs to induce TH1-skewed T cells that persist into old age and eliminate implantable tumors. These results raise the possibility of correcting age-associated immune defects through DC manipulation.

immunology↗

bioRxiv2022

Prioritized single-cell proteomics reveals molecular and functional polarization across primary macrophages

Major aims of single-cell proteomics include increasing the consistency, sensitivity, and depth of protein quantification, especially for proteins and modifications of biological interest. To simultaneously advance all these aims, we developed prioritized Single Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while analyzing identifiable peptides at full duty-cycle, thus increasing proteome depth. These strategies increased the sensitivity, data completeness, and proteome coverage over 2-fold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport. This protein covariation within a treatment condition was similar across the treatment conditions and coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at: scp.slavovlab.net/pSCoPE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/484655v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@c44f04org.highwire.dtl.DTLVardef@1fb3e8org.highwire.dtl.DTLVardef@15c456eorg.highwire.dtl.DTLVardef@d271e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Type III interferons induce pyroptosis in gut epithelial cells and delay tissue restitution upon acute intestinal injury.

Tissue damage and repair are hallmarks of the inflammatory process. Despite a wealth of information focused on the mechanisms that govern tissue damage, mechanistic insight on how inflammatory immune mediators affect the restitution phase is lacking. Here, we investigated how interferons influence tissue restitution after damage of the intestinal mucosa driven by inflammatory or physical injury. We found that type III, but not type I, interferons serve a central role in the restitution process. Type III interferons induce the upregulation of ZBP1, caspase activation, and cleavage of gasdermin C, and drive epithelial cell death by pyroptosis, thus delaying tissue restitution. We also found that this pathway is transcriptionally regulated in IBD patients. Our findings highlight a new molecular signaling cascade initiated by the immune system that affects the outcome of the immune response by delaying tissue repair and that may have important implications for human inflammatory disorders.

immunology↗

bioRxiv2021

An adjuvanted SARS-CoV-2 RBD nanoparticle elicits neutralizing antibodies and fully protective immunity in aged mice

Development of affordable and effective vaccines that can also protect vulnerable populations such as the elderly from COVID-19-related morbidity and mortality is a public health priority. Here we took a systematic and iterative approach by testing several SARS-CoV-2 protein antigens and adjuvants to identify a combination that elicits neutralizing antibodies and protection in young and aged mice. In particular, SARS-CoV-2 receptorbinding domain (RBD) displayed as a protein nanoparticle (RBD-NP) was a highly effective antigen, and when formulated with an oil-in-water emulsion containing Carbohydrate fatty acid MonoSulphate derivative (CMS) induced the highest levels of cross-neutralizing antibodies compared to other oil-in-water emulsions or AS01B. Mechanistically, CMS induced antigen retention in the draining lymph node (dLN) and expression of cytokines, chemokines and type I interferon-stimulated genes at both injection site and dLN. Overall, CMS:RBD-NP is effective across multiple age groups and is an exemplar of a SARS-CoV-2 subunit vaccine tailored to the elderly.

immunology↗

bioRxiv2021

An integrated pipeline and multi-model graphical user interface for accurate nano-dosimetry

Accurate dosing of nanoparticles is crucial for risk assessment and for their safe use in medical and other applications. Although it is well-known that nanoparticles sediment, diffuse and aggregate as they move through a fluid, and that therefore the effective dose perceived by cells may not necessarily be that initially administered, dose quantification remains a challenge. This is because to date, methods for accurate dose estimation are difficult to implement, involving precise characterization of the nanomaterial and the exposure system as well as complex mathematical operations. Here we present a pipeline for accurate nano-dosimetry of engineered nanoparticles on cell monolayers, based on an easy-to-use graphical software - DosiGUI - which integrates two well-established particokinetic and particodynamic models. DosiGUI is an open source tool which was developed to facilitate nano-dosimetrics. The pipeline includes methods for determining the stickiness index which describes the affinity between nanoparticles and cells. Our results show that accurate estimations of the effective dose cannot prescind from rigorous characterization of the stickiness index, which depends on both nanoparticle characteristics and cell type.

pharmacology and toxicology↗

bioRxiv2021

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups ([~]80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. One Sentence SummaryAlum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

immunology↗

bioRxiv2021

Severity of SARS-CoV-2 infection as a function of the interferon landscape across the respiratory tract of COVID-19 patients.

The COVID-19 outbreak driven by SARS-CoV-2 has caused more than 2.5 million deaths globally, with the most severe cases characterized by over-exuberant production of immune-mediators, the nature of which is not fully understood. Interferons of the type I (IFN-I) or type III (IFN-III) families are potent antivirals, but their role in COVID-19 remains debated. Our analysis of gene and protein expression along the respiratory tract shows that IFNs, especially IFN-III, are over-represented in the lower airways of patients with severe COVID-19, while high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity; also, IFN expression varies with abundance of the cell types that produce them. Our data point to a dynamic process of inter- and intra-family production of IFNs in COVID-19, and suggest that IFNs play opposing roles at distinct anatomical sites.

immunology↗

bioRxiv2020

Type III interferons disrupt the lung epithelial barrier upon viral recognition.

Lower respiratory tract infections are a leading cause of mortality driven by infectious agents. RNA viruses such as influenza virus, respiratory syncytial virus and the new pandemic coronavirus SARS-CoV-2 can be highly pathogenic. Clinical and experimental evidence indicate that most severe and lethal cases do not depend on the viral burden and are, instead, characterized by an aberrant immune response. In this work we assessed how the innate immune response contributes to the pathogenesis of RNA virus infections. We demonstrate that type III interferons produced by dendritic cells in the lung in response to viral recognition cause barrier damage and compromise the host tissue tolerance. In particular, type III interferons inhibit tissue repair and lung epithelial cell proliferation, causing susceptibility to lethal bacterial superinfections. Overall, our data give a strong mandate to rethink the pathophysiological roles of this group of interferons and their possible use in the clinical practice against endemic as well as emerging viral infections.

immunology↗

bioRxiv2020

The physical form of microbial ligands bypasses the need for dendritic cell migration to stimulate adaptive immunity

A central paradigm of immunology is that the innate immune system first detects infectious agents in peripheral tissues, shortly after a pathogen has breached an epithelial barrier. This detection event is mediated by pattern recognition receptors in phagocytes, which then migrate to draining lymph nodes (dLNs), where information of a microbial encounter is conveyed to T and B lymphocytes to generate adaptive immunity. Through the study of fungal moieties, we present data that challenge this model. We found that soluble fungal polysaccharides are immunosilent in the periphery, but become potent immunogens in the dLN. These ligands completely bypass the need of phagocyte migration and, instead, directly activate an immune response that is most similar to those that typify viral infections. These data establish a class of microbial products that violate a central tenet of the immunological lexicon and illustrate that the physical form (not just the chemical structure) impacts innate and adaptive immunity.

immunology↗

bioRxiv2018

Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence

Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.

microbiology↗