Gabriele Pollara

Urinary tract infection (UTI) remains a major global health burden, with frequent recurrences and rising antimicrobial resistance compromising treatment efficacy. Conventional susceptibility assays often fail to predict clinical outcomes, highlighting the need for more physiologically relevant infection models. Here, we investigated how microenvironmental complexity influences uropathogenic Escherichia coli (UPEC) responses to antibiotics and bacteriophages using human urine, a three-dimensional human urothelial microtissue model (3D-UHU), and a novel mesofluidic platform (P-FLO) that applies physiologically relevant wall shear stress to 3D-UHU. Nitrofurantoin showed the greatest potency in conventional susceptibility assays but did not eradicate infection in the static 3D-UHU model. A bacteriophage cocktail (LCPR1) inhibited intracellular bacterial communities, preserved urothelial viability and induced inflammatory cytokine and chemokine secretion. Combined LCPR1 + nitrofurantoin treatment eliminated planktonic bacteria and reduced extracellular and intracellular bacterial communities under static conditions but did not further reduce urothelium-associated bacterial burden compared with nitrofurantoin alone. Under flow, shear stress promoted bacterial elongation, attachment and intracellular community formation, while reducing nitrofurantoin and combination therapy efficacy despite increased drug exposure. These findings demonstrate that the bladder microenvironment profoundly influences UPEC infection dynamics and therapeutic outcomes, underscoring the need for advanced models to guide treatment strategies in the antibiotic resistance era.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet therapeutics are lacking. The aim of this study was to develop a pre‑clinical model that recapitulates key clinical outcomes in infants with RSV bronchiolitis, such as neutrophil activation and migration into the airways. Peripheral blood neutrophils from infants with severe RSV disease admitted to the Paediatric Intensive Care Unit showed elevated myeloperoxidase (MPO) in children with RSV, compared to age-matched controls. To mechanistically model this response, we established an air-liquid interface (ALI) system incorporating paediatric airway epithelial cells, endothelial cells and neutrophils from adults, to recapitulate the blood-airway barrier. Following RSV infection, with and without treatment with antivirals remdesivir or RSV604, neutrophil migration and activation were assessed using flow cytometry. While both drugs reduced viral load, only RSV604 attenuated MPO expression. This model suggests that MPO could be useful as a readout of therapeutic efficacy. Targeting neutrophil-driven inflammatory pathways may be critical for reducing pathology in infant RSV infection.

The number 17 is considered unlucky by some Italians as its Roman numerals – XVII – can be rearranged as Vixi , a Latin expression that can be interpreted as “my life is over”. In other contexts, the number 17 is associated with wisdom and success. This paradox holds true when it comes to the role of interleukin 17 (IL-17) in tuberculosis (TB). On one hand, immune correlates of protection studies have consistently identified IL-17 responses as key players in natural and vaccine induced protection against infection and disease. On the other, IL-17 has been proposed as a main driver of the immunopathology that underlies TB morbidity and mortality. Thus, while some researchers seek to develop novel TB vaccine approaches that promote IL-17 responses, others hunt for host-directed therapeutic approaches that block its activity. In this article, we attempt to address this conundrum and synthesise the main arguments supporting a role for IL-17 in the protection and pathogenesis of this deadly human infectious disease. Ultimately, as with superstition, whether the 17th interleukin is friend or foe in human TB is likely to depend on the context.

Background: Nasopharyngeal colonization by Streptococcus pneumoniae is characterized by bacterial adherence to epithelial cells, microinvasion, and innate immune activation. Previously, we have shown that two serotype 6B S pneumoniae mutant strains affecting bacterial metabolism (ΔproABC/pia and Δfhs/pia) colonize humans and mice, but in a murine disease model do not cause invasive infection. Methods: Using an experimental human pneumococcal challenge model, ex vivo airway cells, and in vitro nasopharyngeal epithelium, we explore whether microinvasion and innate immune responses persist despite disease attenuation. Results: We show that under serum stress, these biosynthesis gene mutations had a broad but different impact on pneumococcal virulence gene expression, oxidative stress regulation, and purine and carbohydrate metabolism genes. However, although these mutations did not attenuate microinvasion in human challenge and epithelial models, there was less transmigration of Detroit 562 nasopharyngeal epithelial cells by the mutants compared to wild-type. Cellular reorganization of primary human airway epithelium varied considerably between strains. Compared to wild-type, infection of Detroit 562 epithelial cells by the Δfhs/piaA strain, but not the ΔproABC/piaA strain, was less proinflammatory, induced less caspase 8 production, and was associated with increased pneumococcal hydrogen peroxide and reduced pneumolysin secretion. Conclusions: These findings suggest that differences in microinvasion and epithelial responses were driven by the differential expression of multiple bacterial virulence and metabolic pathways. These data highlight the complex impact of single gene mutations on bacterial virulence and suggest that the virulence determinants of pneumococcal epithelial colonization, microinvasion, and innate immunity are not necessarily directly linked to disease.

Background Antimicrobial stewardship (AMS) aims to de-escalate antibiotics from broad to narrow spectrum but there is a lack of evidence to support selection of patients who would most benefit from AMS review. Objectives We aimed to compare the days of antibiotic spectrum coverage (DASC) score with a standard AMS metric (days of therapy, DOT), to evaluate DASC as a tool to measure AMS impact. Methods Multi-centre retrospective cohort study of medical and surgical patients in two London hospitals. DASC and DOT were calculated for each prescription before and after AMS intervention and change in DOT and DASC calculated. We assessed correlation between DOT and DASC scores and associations between patient factors and reduction in post-AMS DOT or DASC. Results We evaluated 1287 prescriptions in 789 patients. AMS-associated change in DOT and DASC were correlated, but there was still substantial discordance between changes in DASC and changes in DOT, indicating that DASC may yield greater resolution on the impact of AMS review than DOT. Patients’ cumulative antibiotic spectrum score from all antibiotic prescriptions prior to AMS review correlated with the ensuing changes in DASC but not with changes in DOT. Conclusions Our findings support further assessment of the standardised antibiotic spectrum metric, DASC, for use in hospital AMS programmes to minimise adverse consequences of broad-spectrum antibiotic prescribing. Implementation of antibiotic spectrum measures have the potential to more accurately quantify the beneficial impact of inpatient AMS reviews, and assist in prioritising patients that most benefit from AMS-led interventions.

T cells contribute to immune protection and pathogenesis in tuberculosis, but measurements of polyclonal responses have failed to resolve correlates of outcome. We report the temporal evaluation of the human in vivo clonal repertoire of Mycobacterium tuberculosis ( Mtb )-reactive T cell responses, by T cell receptor (TCR) sequencing at the site of the tuberculin skin test, as a model for a standardised antigenic challenge. Initial non-selective recruitment of T cells is followed by enrichment of Mtb -reactive clones arising from oligoclonal T cell proliferation. We introduce a modular computational pipeline, Metaclonotypist, to sensitively cluster distinct TCRs with shared epitope specificity, which we apply here to establish a catalogue of public Mtb -reactive HLA-restricted T cell metaclones. Although most in vivo Mtb -reactive T cells are private, 10 metaclones were sufficient to identify Mtb -T cell reactivity across our study population (N≥128), indicating striking population level immunodominance of specific TCR-peptide interactions that may inform patient stratification and vaccine development.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet effective therapeutics are lacking. The aim of this study is to develop an in vitro model that recapitulates key clinical outcomes in infants with RSV bronchiolitis to help accelerate the discovery of effective therapeutics. Neutrophil activation and influx into the airways are hallmarks of severe RSV infection, but these responses are difficult to quantify in clinical trials. Here we profile peripheral blood-derived neutrophils from infants with RSV admitted to the Paediatric Intensive Care Unit (PICU) and identify myeloperoxidase (MPO) as a key indicator of disease severity when compared to age matched controls. To mechanistically model this response, we established a paediatric airway epithelial air–liquid interface (ALI) system incorporating an endothelial layer and primary neutrophils to recapitulate the tissue microenvironment at the blood-airway barrier. Following RSV infection, neutrophil migration and activation were assessed using flow cytometry. The inclusion of an endothelial layer enhanced physiological relevance and more accurately replicated in vivo MPO responses. We then evaluated two antiviral candidates (remdesivir (RDV) and RSV604) to assess their ability to modulate neutrophil activation. While both compounds reduced viral load at 24 hours post-infection, only RSV604 attenuated MPO expression. These findings establish MPO as both a biomarker of RSV disease severity and a functional readout of therapeutic efficacy and demonstrate that targeting neutrophil⍰driven inflammatory pathways may be critical for reducing pathology in infant RSV infection. Take home message: Antiviral drug discovery should include neutrophil MPO reduction as a readout of therapeutic efficacy. Graphical

Type I interferon responses have been considered detrimental to host protection in tuberculosis (TB). We provide novel data to challenge this paradigm, derived from transcriptional profiling of human in vivo immune responses to discover associations with radiographic disease severity in pulmonary TB, combined with mechanistic studies to test causality for observed associations using a zebrafish larval mycobacterial infection model. Type I interferon activity in tissue samples from the site of a standardised mycobacterial challenge, the tuberculin skin test, was associated with less severe human TB disease. Abrogation of type I interferon signalling, by CRISPR-mediated mutagenesis of stat2, led to increased burden and dissemination of Mycobacterium marinum infection in zebrafish larvae. The mechanism for increased severity of mycobacterial infection in zebrafish involves reduced recruitment of myeloid cells required to restrict bacterial growth. Our data support a clear host protective role for type I interferon responses in mycobacterial infection, with potential applications for risk-stratification of adverse outcomes and development of a host-directed therapy to mitigate against severe disease.

T cells contribute to immune protection and pathogenesis in tuberculosis, but measurements of polyclonal responses have failed to resolve correlates of outcome. We report the first temporal evaluation of the human in vivo clonal repertoire of Mtb-reactive T cell responses, by T cell receptor (TCR) sequencing at the site of a standardised antigenic challenge. Initial recruitment of non-Mtb reactive T cells is followed by enrichment of Mtb-reactive clones arising from oligoclonal T cell proliferation. We introduce a modular computational pipeline, Metaclonotypist, to sensitively cluster distinct TCRs with shared epitope specificity, which we apply here to establish a catalogue of public Mtb-reactive HLA-restricted T cell metaclones. Although most in vivo Mtb-reactive T cells are private, 10 metaclones were sufficient to identify Mtb-T cell reactivity across our study population (N≥128), indicating striking population level immunodominance of specific TCR-peptide interactions that may offer novel approaches to patient stratification and vaccine development.