Biology subjects

Moschetti, G.

Publications and source records attributed to Moschetti, G..

4 recordsLinked to original sources

bioRxiv2025

A vesicle-based platform for high-efficiency, high-viability CRISPR/Cas9 knockout in primary human myeloid cells

Myeloid cells, including monocytes, macrophages, and dendritic cells, are central to host defence, inflammation, and antigen presentation. However, functional genetics studies in corresponding primary cells remain limited due to inefficient delivery methods. Here we report a vesicle-based CRISPR/Cas9 platform for effective genome editing in primary human myeloid cells using engineered virus-like particles (VLPs) and extracellular vesicles (EVs) to deliver Cas9-gRNA ribonucleoproteins (RNPs). Targeting primary CD14+ monocytes enables rapid generation of robust polyclonal knockouts while preserving cell viability and subsequent differentiation into macrophages and dendritic cells. Because our workflow relies on standard cell-culture handling rather than electroporation instrumentation, it is inexpensive, highly scalable and readily transferable across laboratories. Edited cells remain compatible with diverse downstream assays and functional readouts, enabling scalable loss-of-function studies and mechanistic dissection of primary human myeloid cell biology in health and disease. Vesicle-mediated RNP delivery thus provides a broadly applicable route to genetic perturbation in primary human myeloid lineages. SummaryFiori and colleagues describe a vesicle-based platform for efficient CRISPR/Cas9 knockout in primary human myeloid cells. This approach preserves cell viability, enables near-complete gene disruption, and supports functional studies of innate immunity, antigen presentation, and HIV-1 infection.

immunology↗

bioRxiv2024

Chronic mycobacteria infection triggers macrophage senescence

Chronic infections with intracellular pathogens such as Mycobacterium abscessus (Mab) pose significant health challenges due to their capacity to persist within host cells and evade immune responses. This study investigates the cellular responses to chronic Mab infection in macrophages, particularly focusing on cellular senescence. Using an in vitro model of chronic infection in murine alveolar-like macrophages, we found that Mab induces a senescent phenotype characterised by decreased proliferation, altered morphology, DNA damage signalling activation, and upregulation of senescence markers such as p21 and SA-{beta}-galactosidase. Intriguingly, senescent macrophages secreted pro-inflammatory cytokines, consistent with a senescence-associated secretory phenotype (SASP), which promoted secondary senescence in neighbouring uninfected cells. This paracrine transmission of senescence underscores a potentially deleterious effect of Mab-induced SASP on tissue microenvironments, fostering a pro-inflammatory niche that may contribute to pathogen persistence. These findings highlight Mab-induced senescence as a key factor in chronic infection pathology, suggesting that targeting senescent cells and SASP-related pathways could enhance treatment outcomes in chronic bacterial infections.

microbiology↗

bioRxiv2024

Autosomal Dominant-Hyper-IgE Syndrome patients contain pre-Th17-cells that are activated by opportunistic pathogens to produce IL-10

BackgroundAutosomal Dominant-Hyper-IgE Syndrome (AD-HIES) is caused by dominant-negative (DN) STAT3 mutations and characterized by high IgE levels, a lack of Th17-cells and recurrent infections with extracellular pathogens. We previously identified an enigmatic population of IL-10 producing CCR6+B-helper T-cells and investigated here their relationship to Th17-cells and STAT3 signalling requirements. MethodsHuman blood lymphocytes were analysed by multiparametric flow cytometry in healthy donors and AD-HIES patients. Analysis was performed by conventional gating or with bioinformatic tools. FACS-purified T-cell subsets were activated in vitro and Th17 differentiation assessed. T-cell antigen specificities were assessed by activation with heat-killed pathogens or antigenic peptide pools. B helper capacities were determined according to antibody secretion in B-T co-cultures by ELISA. ResultsCCR6+Th-cells that lacked subset-defining differentiation markers were mostly non-polarised central memory T-cells (TCM) that produced IL-10 and expressed ROR{gamma}t. They were pre-committed to a Th17 fate, since TCR stimulation in the absence of polarising cytokines induced efficient Th17 differentiation. The latter was promoted by an autocrine loop of STAT3-activating cytokines. CCR6+Th-cells were reduced in patients with DN-STAT3 mutations but contained activated CCR6+TCM that produced IL-10 and responded vigorously to AD-HIES-associated pathogens. These residual CCR6+Th-cells provided B-cell help for IgG and IgE production. ConclusionsTh17 differentiation in AD-HIES patients was not completely impaired but arrested at an intermediate stage of IL-10 producing "pre-Th17"-cells. Surprisingly, DN-STAT3 mutations did not inhibit IL-10 production by CD4+T-cells. Pre-Th17-cells were activated by AD-HIES-associated pathogens and possessed B-helper functions, suggesting that they are not protective but promote aberrant IgE production.

immunology↗

bioRxiv2023

Endolysin B: A new archetype in M. tuberculosis treatment

So far it is thanks to antibiotics that illnesses, such as Tuberculosis (TB), are treatable. However, antimicrobial misuse combined with Mycobacterium tuberculosis phenotypic plasticity are nullifying the effects of the existing therapies. As a result, increasingly people are dying (one person dies of TB every 20 seconds), especially due to the rise of multi-and extensively drug-resistant strains. There is indeed a urgent need for new and more effective therapies, which should match the requirement of avoiding the rise in drug resistance. Among them, the use of bacteriophage - bacteria-restricted viruses - or simply phage lytic enzyme (i.e., endolysin) represents one of the most promising alternatives. All phages encode for the endolysin A (LysA), which degrades the bacteria cell wall, finally leading to the release of the newly synthesized virions. Nevertheless, mycobacteriophages (bacterial viruses selectively infecting mycobacteria), evolved the additional endolysin B (LysB) to selectively damage the complex mycobacteria cell wall, and to evade from their host. LysB, owing a lipolytic enzyme, can degrade the thick mycolic acid layer, and hence disrupt the integrity of the mycobacterial membrane. Despite its key role in mediating mycobacteria lysis, the molecular mechanism regulating LysB binding to its target remains poorly characterized. Herein, we selected Ms6LysB and created a fluorescent engineered version as a proxy to analyze LysB binding qualitatively and quantitatively to both the fast-growing non-pathogenic Mycobacterium smegmatis and the slow-growing pathogenic M. tuberculosis. Additionally, we shed light on LysB antimicrobial activity upon M. tuberculosis infection, by using alveolar-like mouse macrophages (mAMs) as a cellular model that closely recapitulates the natural niche of M. tuberculosis infection. Our study provides the proof-of-principle that Ms6 LysB binding to the outer mycobacterial membrane can impair M. tuberculosis growth homeostasis and that LysB retain its lytic properties even when internalized by mAMs. This lays the groundwork for the use of LysB as a new therapeutic strategy to undermine M. tuberculosis infection.

microbiology↗