Many biological pathways were first uncovered by identifying mutants with visible phenotypes and by scoring every sample in a screen via tedious and subjective visual inspection. Now, automated image analysis can effectively score many phenotypes. In practical application, customizing an image-analysis algorithm or finding a sufficient number of example cells to train a machine learning algorithm can be infeasible, particularly when positive control samples are not available and the phenotype of interest is rare. Here we present a supervised machine learning approach that uses iterative feedback to readily score multiple subtle and complex morphological phenotypes in high-throughput, image-based screens. First, automated cytological profiling extracts hundreds of numerical descriptors for every cell in every image. Next, the researcher generates a rule (i.e., classifier) to recognize cells with a phenotype of interest during a short, interactive training session using iterative feedback. Finally, all of the cells in the experiment are automatically classified and each sample is scored based on the presence of cells displaying the phenotype. By using this approach, we successfully scored images in RNA interference screens in 2 organisms for the prevalence of 15 diverse cellular morphologies, some of which were previously intractable.high-content screening ͉ high-throughput image analysis ͉ phenotype T he history of biology has been dramatically shaped by classic visual screens in model organisms, including Drosophila melanogaster (1-3), Saccharomyces cerevisiae (4), Caenorhabditis elegans (5), and the zebrafish Danio rerio (6, 7). In each case, biological pathways were discovered because researchers were intrigued by groups of peculiar-looking mutants and identified the genes underlying their phenotypes. Because researchers have favored the extensive study of relatively few genes (8), classic, wide-net approaches like screening are as relevant as ever to probe known biological pathways and discover new ones. Modern technology now enables large-scale experiments in cultured cells to identify human genes that underlie biological processes via RNAi. Automation also allows the screening of chemical libraries to identify perturbants useful as research tools or drugs.Despite these advances, scoring cells in images for rare and unusual morphologies has, in general, remained a significant bottleneck (9-12). Cell image analysis allows accurate identification and measurement of cells' features, enabling automated analysis of certain phenotypes that were previously intractable (13-26). However, many interesting phenotypes require the assessment of several measured features of cells. Machine learning methods that select and combine multiple features for automated cell classification have been used to score many phenotypes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). These methods require the provision of example cells that do and do not display the morphology of interest (i.e., positive and negative cells). Finding posi...

Stressors ranging from nutrient deprivation to immune signaling can induce the degradation of cytoplasmic material by a process known as autophagy. Increasingly, research on autophagy has begun to focus on its role in inflammation and the immune response. Autophagy acts as an immune effector that mediates pathogen clearance. The roles of autophagy bridge both the innate and adaptive immune systems and include functions in thymic selection, antigen presentation, promotion of lymphocyte homeostasis and survival, and regulation of cytokine production. In this review, we discuss the mechanisms by which autophagy is regulated, as well as the functions of autophagy and autophagy proteins in immunity and inflammation.

Background & Aims Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting anti-microbial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. Methods We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1f/f x Villin-cre) or CD11c+ cells (Atg16l1f/f x CD11c-cre); these mice were used to assess cell type-specific, anti-bacterial autophagy. All responses were compared to Atg16l1f/f mice (controls). Mice were infected with Salmonella enterica serovar Typhimurium; cecum and small intestine tissues were collected for immunofluorescence, histology, and quantitative reverse transcription PCR analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on anti-bacterial responses in human epithelial cells. Results Autophagy was induced in small intestine and cecum following infection with S Typhimurium, and required Atg16l1. S Typhimurium colocalized with microtubule-associated protein 1 light chain 3 beta (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1f/f x Villin-cre mice. Atg16l1f/f x Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMP. Consistent with these defective immune responses, Atg16l1f/f x Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1f/f x CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. Conclusions Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It is also required to prevent systemic infection of mice with enteric bacteria.

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.

Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK) related kinase TBK1 in vivo, associated with increased neutrophil and T cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma (PDAC) cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro. Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKε/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting.

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate Ϸ100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles.

Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways, and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1β production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naïve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1β production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1β, and to test novel therapeutic hypotheses.

NA interference (RNAi) therapeutics use an endogenous mechanism whereby short interfering RNAs (siRNAs) direct the RNA-induced silencing complex (RISC) to sequence matched target transcripts for knockdown 1 . Both lipid nanoparticles and N-acetylgalactosamine (GalNAc) conjugates are clinically validated and approved delivery strategies for liver targets [2][3][4][5][6][7][8] . Building on nearly 2 decades of siRNA design and chemistry optimization [9][10][11][12] , we demonstrate here that, with suitable delivery solutions, the RNAi pathway can be harnessed in extrahepatic tissues, such as the central nervous system (CNS), eye and lung. Multiple CNS diseases, representing some of the highest unmet medical needs and greatest therapeutic challenges, have been associated with dominant gain-of-function mutations, making them suitable candidates for an RNAi-based silencing approach. As such, chemically modified siRNAs have demonstrated potent and sustained silencing in rodents and non-human primates (NHPs); however, using an invasive intracerebroventricular (ICV) administration approach 13 that is not suitable for repeated dosing in humans. Furthermore, technologies enabling siRNA delivery across the blood-brain barrier following a less challenging systemic administration are similarly being explored [14][15][16][17] , which are, however, still in early stages of discovery. In the eye, intravitreal (IVT) dosing of siRNAs has been evaluated in late-stage clinical studies, with few safety concerns, but did not advance further due to lack of efficacy 18 . Recently, the Coronavirus Disease 2019 (COVID-19) pandemic has highlighted the importance of optimizing siRNA delivery to the lung for the treatment of emergent viral respiratory diseases. Although earlier programs have already shown potential clinical benefits of siRNA-based therapeutics in the lung 19 , 2′-O-hexadecyl (C16) conjugates demonstrate enhanced delivery and siRNA uptake into the alveolar and bronchiolar epithelium. Taken together, this work highlights that the combination of a C16 lipophilic modification with our fully chemically modified, metabolically stable siRNAs achieves efficient delivery to the CNS, eye and lung, resulting in a robust and durable gene silencing in rodents and NHPs, with a favorable safety profile. We think that these advances have the potential to generate multiple candidates for investigating clinical safety and efficacy in humans. ResultsOptimization of the siRNA conjugate design. Lipophilic moieties represent one of the earliest approaches to improve cellular uptake and delivery of antisense oligonucleotides (ASOs) and siRNAs to the liver and various other organ systems 20 , including the CNS [21][22][23] . We reasoned that, by carefully optimizing the lipophilicity of chemically modified siRNAs, we could enhance intracellular delivery without compromising broad biodistribution, potency and safety. We used the 2′ position of the ribose sugar backbone to introduce