An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.

Group 2 innate lymphoid cells constrain type 3/17 lymphocytes in shared stromal niches to restrict liver fibrosis.

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5 + lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33 high niche fibroblasts. Ablation of IL-5 + lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A + type 3/17 lymphocytes, predominantly γδ T cells. In contrast, concurrent ablation of IL-5 + and IL-17A + lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

Sentinel p16INK4a+ cells in the basement membrane form a reparative niche in the lung.

We engineered an ultrasensitive reporter of p16INK4a, a biomarker of cellular senescence. Our reporter detected p16INK4a-expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.

Workshop-based learning and networking: a scalable model for research capacity strengthening in low- and middle-income countries.

Science education and research have the potential to drive profound change in low- and middle-income countries (LMICs) through encouraging innovation, attracting industry, and creating job opportunities. However, in LMICs, research capacity is often limited, and acquisition of funding and access to state-of-the-art technologies is challenging. The Alliance for Global Health and Science (the Alliance) was founded as a partnership between the University of California, Berkeley (USA) and Makerere University (Uganda), with the goal of strengthening Makerere University's capacity for bioscience research. The flagship program of the Alliance partnership is the MU/UCB Biosciences Training Program, an in-country, hands-on workshop model that trains a large number of students from Makerere University in infectious disease and molecular biology research. This approach nucleates training of larger and more diverse groups of students, development of mentoring and bi-directional research partnerships, and support of the local economy. Here, we describe the project, its conception, implementation, challenges, and outcomes of bioscience research workshops. We aim to provide a blueprint for workshop implementation, and create a valuable resource for bioscience research capacity strengthening in LMICs.

Development of a Dual-Fluorescent-Reporter System in Clostridioides difficile Reveals a Division of Labor between Virulence and Transmission Gene Expression.

The bacterial pathogen Clostridioides difficile causes gastroenteritis by producing toxins and transmits disease by making resistant spores. Toxin and spore production are energy-expensive processes that are regulated by multiple transcription factors in response to many environmental inputs. While toxin and sporulation genes are both induced in only a subset of C. difficile cells, the relationship between these two subpopulations remains unclear. To address whether C. difficile coordinates the generation of these subpopulations, we developed a dual-transcriptional-reporter system that allows toxin and sporulation gene expression to be simultaneously visualized at the single-cell level using chromosomally encoded mScarlet and mNeonGreen fluorescent transcriptional reporters. We then adapted an automated image analysis pipeline to quantify toxin and sporulation gene expression in thousands of individual cells under different medium conditions and in different genetic backgrounds. These analyses revealed that toxin and sporulation gene expression rarely overlap during growth on agar plates, whereas broth culture increases this overlap. Our results suggest that certain growth conditions promote a "division of labor" between transmission and virulence gene expression, highlighting how environmental inputs influence these subpopulations. Our data further suggest that the RstA transcriptional regulator skews the population to activate sporulation genes rather than toxin genes. Given that recent work has revealed population-wide heterogeneity for numerous cellular processes in C. difficile, we anticipate that our dual-reporter system will be broadly useful for determining the overlap between these subpopulations. IMPORTANCE Clostridioides difficile is an important nosocomial pathogen that causes severe diarrhea by producing toxins and transmits disease by producing spores. While both processes are crucial for C. difficile disease, only a subset of cells express toxins and/or undergo sporulation. Whether C. difficile coordinates the subset of cells inducing these energy-expensive processes remains unknown. To address this question, we developed a dual-fluorescent-reporter system coupled with an automated image analysis pipeline to rapidly compare the expression of two genes of interest across thousands of cells. Using this system, we discovered that certain growth conditions, particularly growth on agar plates, induce a "division of labor" between toxin and sporulation gene expression. Since C. difficile exhibits phenotypic heterogeneity for numerous vital cellular processes, this novel dual-reporter system will enable future studies aimed at understanding how C. difficile coordinates various subpopulations throughout its infectious disease cycle.

TLR2 and endosomal TLR-mediated secretion of IL-10 and immune suppression in response to phagosome-confined Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.