The alarming diffusion of multidrug-resistant (MDR) bacterial strains requires investigations on nonantibiotic therapies. Among such therapies, the use of bacteriophages (phages) as antimicrobial agents, namely, phage therapy, is a promising treatment strategy supported by the findings of recent successful compassionate treatments in Europe and the United States. In this work, we combined host range and genomic information to design a 6-phage cocktail killing several clinical strains ofPseudomonas aeruginosa, including those collected from Italian cystic fibrosis (CF) patients, and analyzed the cocktail performance. We demonstrated that the cocktail composed of four novel phages (PYO2, DEV, E215 and E217) and two previously characterized phages (PAK_P1 and PAK_P4) was able to lyseP. aeruginosaboth in planktonic liquid cultures and in biofilms. In addition, we showed that the phage cocktail could cure acute respiratory infection in mice and treat bacteremia in wax moth (Galleria mellonella) larvae. Furthermore, administration of the cocktail to larvae prior to bacterial infection provided prophylaxis. In this regard, the efficiency of the phage cocktail was found to be unaffected by the MDR or mucoid phenotype of the pseudomonal strain. The cocktail was found to be superior to the individual phages in destroying biofilms and providing a faster treatment in mice. We also found theGallerialarva model to be cost-effective for testing the susceptibility of clinical strains to phages, suggesting that it could be implemented in the frame of developing personalized phage therapies.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

Phagotherapy, the use of bacteriophages to fight bacterial infections as an alternative to antibiotic treatments, has become of increasing interest in the last years. This is mainly due to the diffusion of multi-drug resistant (MDR) bacterial infections that constitute a serious issue for public health. Phage therapy is gaining favor due to its success in agriculture and veterinary treatments and its extensive utilization for human therapeutic protocols in the Eastern world. In the last decades, some clinical trials and compassionate treatments have also been performed in the Western world, indicating that phage therapy is getting closer to its introduction in standard therapy protocols. However, several questions concerning the use of phages in human therapeutic treatments are still present and need to be addressed. In this review, we illustrate the state of art of phage therapy and examine the role of animal models to translate these treatments to humans.

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMC^PCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMC^PCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMC^PCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMC^PCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.

Extracellular vesicles (EVs) are important components of the metastatic niche and are crucial in infiltration, metastasis, and immune tolerance processes during tumorigenesis. We hypothesized that human endogenous retroviruses (HERV) positive EVs derived from tumor cellsmay have a role in modulating the innate immune response. The study was conducted in two different colorectal cancer cell lines, representing different stages of cancer development: Caco-2, derived from a non-metastatic colorectal adenocarcinoma, and SK-CO-1, derived from metastatic colorectal adenocarcinoma (ascites). Both cell lines were treated with decitabine to induce global hypomethylation and to reactivate HERV expression. EVs were quantified by nanoparticle tracking analysis, and HERV-positive EV concentrations were measured by flow cytometry. The effect of EVs isolated from both untreated and decitabine-treated cells on the innate immune response was evaluated by injecting them in zebrafish embryos and then assessing Interleukin 1β (IL1-β), Interleukin 10 (IL-10), and the myeloperoxidase (mpx) expression levels by real-time qPCR. Interestingly, HERV-K positive EVs concentrations were significantly associated with a reduced expression of IL1-β and mpx, supporting our hypothesis that HERV-positive EVs may act as immunomodulators in tumor progression. The obtained results open new perspectives about the modulation of the immune response in cancer therapy.

Short-term exposure to fine particulate matter (PM_2.5) is associated with the activation of adverse inflammatory responses, increasing the risk of developing acute respiratory diseases, such as those caused by pathogen infections. However, the functional mechanisms underlying this evidence remain unclear. In the present study, we generated a zebrafish model of short-term exposure to a specific PM_2.5, collected in the northern metropolitan area of Milan, Italy. First, we assessed the immunomodulatory effects of short-term PM_2.5 exposure and observed that it elicited pro-inflammatory effects by inducing the expression of cytokines and triggering hyper-activation of both neutrophil and macrophage cell populations. Moreover, we examined the impact of a secondary infectious pro-inflammatory stimulus induced through the injection of Pseudomonas aeruginosa lipopolysaccharide (Pa-LPS) molecules after exposure to short-term PM_2.5. In this model, we demonstrated that the innate immune response was less responsive to a second pro-inflammatory infectious stimulus. Indeed, larvae exhibited dampened leukocyte activation and impaired production of reactive oxygen species. The obtained results indicate that short-term PM_2.5 exposure alters the immune microenvironment and affects the inflammatory processes, thus potentially weakening the resistance to pathogen infections.