Regulatory module involving FGF13, miR-504, and p53 regulates ribosomal biogenesis and supports cancer cell survival.

The microRNA miR-504 targets TP53 mRNA encoding the p53 tumor suppressor. miR-504 resides within the fibroblast growth factor 13 (FGF13) gene, which is overexpressed in various cancers. We report that the FGF13 locus, comprising FGF13 and miR-504, is transcriptionally repressed by p53, defining an additional negative feedback loop in the p53 network. Furthermore, we show that FGF13 1A is a nucleolar protein that represses ribosomal RNA transcription and attenuates protein synthesis. Importantly, in cancer cells expressing high levels of FGF13, the depletion of FGF13 elicits increased proteostasis stress, associated with the accumulation of reactive oxygen species and apoptosis. Notably, stepwise neoplastic transformation is accompanied by a gradual increase in FGF13 expression and increased dependence on FGF13 for survival ("nononcogene addiction"). Moreover, FGF13 overexpression enables cells to cope more effectively with the stress elicited by oncogenic Ras protein. We propose that, in cells in which activated oncogenes drive excessive protein synthesis, FGF13 may favor survival by maintaining translation rates at a level compatible with the protein quality-control capacity of the cell. Thus, FGF13 may serve as an enabler, allowing cancer cells to evade proteostasis stress triggered by oncogene activation.

Rapid and specific detection of single nanoparticles and viruses in microfluidic laminar flow via confocal fluorescence microscopy.

Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we report the development of a flow virometer for the specific and rapid detection of single nanoparticles based on confocal microscopy. The combination of laminar flow and multiple dyes enable the detection of correlated fluorescence signals, providing information on nanoparticle volumes and specific chemical composition properties, such as viral envelope proteins. We evaluated and validated the assay using fluorescent beads and viruses, including SARS-CoV-2. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting clinically-relevant virus loads. Based on our results, we envision the use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup.

Human Multi-Compartment Airways-on-Chip Platform for Emulating Respiratory Airborne Transmission: From Nose to Pulmonary Acini.

The past decade has witnessed tremendous endeavors to deliver novel preclinical in vitro lung models for pulmonary research endpoints, including foremost with the advent of organ- and lung-on-chips. With growing interest in aerosol transmission and infection of respiratory viruses within a host, most notably the SARS-CoV-2 virus amidst the global COVID-19 pandemic, the importance of crosstalk between the different lung regions (i.e., extra-thoracic, conductive and respiratory), with distinct cellular makeups and physiology, are acknowledged to play an important role in the progression of the disease from the initial onset of infection. In the present Methods article, we designed and fabricated to the best of our knowledge the first multi-compartment human airway-on-chip platform to serve as a preclinical in vitro benchmark underlining regional lung crosstalk for viral infection pathways. Combining microfabrication and 3D printing techniques, our platform mimics key elements of the respiratory system spanning (i) nasal passages that serve as the alleged origin of infections, (ii) the mid-bronchial airway region and (iii) the deep acinar region, distinct with alveolated airways. Crosstalk between the three components was exemplified in various assays. First, viral-load (including SARS-CoV-2) injected into the apical partition of the nasal compartment was detected in distal bronchial and acinar components upon applying physiological airflow across the connected compartment models. Secondly, nebulized viral-like dsRNA, poly I:C aerosols were administered to the nasal apical compartment, transmitted to downstream compartments via respiratory airflows and leading to an elevation in inflammatory cytokine levels secreted by distinct epithelial cells in each respective compartment. Overall, our assays establish an in vitro methodology that supports the hypothesis for viral-laden airflow mediated transmission through the respiratory system cellular landscape. With a keen eye for broader end user applications, we share detailed methodologies for fabricating, assembling, calibrating, and using our multi-compartment platform, including open-source fabrication files. Our platform serves as an early proof-of-concept that can be readily designed and adapted to specific preclinical pulmonary research endpoints.

PEG-fibrinogen hydrogel microspheres as a scaffold for therapeutic delivery of immune cells.

Recent advances in the field of cell therapy have proposed new solutions for tissue repair and regeneration using various cell delivery approaches. Here we studied ex vivo a novel topical delivery system of encapsulated cells in hybrid polyethylene glycol-fibrinogen (PEG-Fb) hydrogel microspheres to respiratory tract models. We investigated basic parameters of cell encapsulation, delivery and release in conditions of inflamed and damaged lungs of bacterial-infected mice. The establishment of each step in the study was essential for the proof of concept. We demonstrated co-encapsulation of alveolar macrophages and epithelial cells that were highly viable and equally distributed inside the microspheres. We found that encapsulated macrophages exposed to bacterial endotoxin lipopolysaccharide preserved high viability and secreted moderate levels of TNFα, whereas non-encapsulated cells exhibited a burst TNFα secretion and reduced viability. LPS-exposed encapsulated macrophages exhibited elongated morphology and out-migration capability from microspheres. Microsphere degradation and cell release in inflamed lung environment was studied ex vivo by the incubation of encapsulated macrophages with lung extracts derived from intranasally infected mice with Yersinia pestis, demonstrating the potential in cell targeting and release in inflamed lungs. Finally, we demonstrated microsphere delivery to a multi-component airways-on-chip platform that mimic human nasal, bronchial and alveolar airways in serially connected compartments. This study demonstrates the feasibility in using hydrogel microspheres as an effective method for topical cell delivery to the lungs in the context of pulmonary damage and the need for tissue repair.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

Distinct extracellular-matrix remodeling events precede symptoms of inflammation.

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases.

COMMD10-Guided Phagolysosomal Maturation Promotes Clearance of Staphylococcus aureus in Macrophages.

Staphylococcus aureus is a major cause of infectious disease. Liver Kupffer cells (KCs) are responsible for sequestering and destroying S. aureus through the phagolysosomal pathway. Proteins belonging to the COMMD family emerge as key intracellular regulators of protein trafficking, but the role of COMMD10 in macrophage-mediated S. aureus eradication is unknown. Here we report that COMMD10 in macrophages was necessary for its timely elimination, as demonstrated with two different S. aureus subspecies. In vivo, COMMD10-deficient liver KCs exhibited impaired clearance of systemic S. aureus infection. S. aureus-infected COMMD10-deficient macrophages exhibited impaired activation of the transcription factor EB, resulting in reduced lysosomal biogenesis. Moreover, S. aureus-initiated phagolysosomal maturation and function were significantly attenuated in COMMD10-deficient macrophages. Finally, expression of COMMD/CCDC22/CCDC93 complex, linked to phagolysosomal maturation, was reduced by COMMD10 deficiency. Collectively, these results support an important role for COMMD10 in instructing macrophage phagolysosomal biogenesis and maturation during S. aureus infection.

Impaired COMMD10-Mediated Regulation of Ly6Chi Monocyte-Driven Inflammation Disrupts Gut Barrier Function.

Ly6Chi monocyte tissue infiltrates play important roles in mediating local inflammation, bacterial elimination and resolution during sepsis and inflammatory bowel disease (IBD). Yet, the immunoregulatory pathways dictating their activity remain poorly understood. COMMD family proteins are emerging as key regulators of signaling and protein trafficking events during inflammation, but the specific role of COMMD10 in governing Ly6Chi monocyte-driven inflammation is unknown. Here we report that COMMD10 curbs canonical and non-canonical inflammasome activity in Ly6Chi monocytes in a model of LPS-induced systemic inflammation. Accordingly, its deficiency in myeloid cells, but not in tissue resident macrophages, resulted in increased Ly6Chi monocyte liver and colonic infiltrates, elevated systemic cytokine storm, increased activation of caspase-1 and-11 in the liver and colon, and augmented IL-1ß production systemically and specifically in LPS-challenged circulating Ly6Chi monocytes. These inflammatory manifestations were accompanied by impaired intestinal barrier function with ensuing bacterial dissemination to the mesenteric lymph nodes and liver leading to increased mortality. The increased inflammasome activity and intestinal barrier leakage were ameliorated by the inducible ablation of COMMD10-deficient Ly6Chi monocytes. In consistence with these results, COMMD10-deficiency in Ly6Chi monocytes, but not in intestinal-resident lamina propria macrophages, led to increased IL-1ß production and aggravated colonic inflammation in a model of DSS-induced colitis. Finally, COMMD10 expression was reduced in Ly6Chi monocytes and their corresponding human CD14hi monocytes sorted from mice subjected to DSS-induced colitis or from IBD patients, respectively. Collectively, these results highlight COMMD10 as a negative regulator of Ly6Chi monocyte inflammasome activity during systemic inflammation and IBD.

Cholinergic Anti-Inflammatory Pathway Does Not Contribute to Prevention of Ulcerative Colitis by Novel Indoline Carbamates.

Indoline carbamates, AN680 and AN917 decrease cytokines, TNF-α and IL-6 in peritoneal macrophages activated by lipopolysaccharide (LPS) and in mouse tissues after LPS injection. They prevent nuclear translocation of nuclear factor κB (NF-κB) and activator protein 1. Only AN917 inhibits cholinesterase (ChE) at relevant concentrations. ChE inhibitors decrease NF-κB by activating α7 nicotinic acetylcholine receptors (α7nAChR). The current study compared the effect of rivastigmine, a ChE inhibitor, AN680 and AN917 on ulcerative colitis induced in mice by ingestion of dextran sodium sulfate (4.5%) solution. Rivastigmine (1 mg/kg), AN680 (2.5-10 mg/kg) and AN917 (2-5 mg/kg) were injected subcutaneously once daily for 8 days. Disease severity was assessed by disease activity index (DAI), colonoscopy, colon length and body weight loss, colonic levels of TNF-α, IL-6, IL-1ß and myeloid peroxidase (MPO) activity. AN680 (5 mg/kg) reduced DAI, colon shrinkage, weight loss, histopathological signs of colon damage, MPO activity, TNF-α, IL-1ß and IL-6 levels without inhibiting ChE. AN917 (5 mg/kg) and rivastigmine (1 mg/kg) inhibited ChE in plasma and colon by 65%, reduced DAI, MPO activity and IL-6, but not TNF-α or IL-1ß. AN917 did not prevent weight loss or colon shrinkage. Mecamylamine abolished the reduction of DAI, MPO activity and IL-6 by AN917 and rivastigmine, indicating they were mediated by α7nAChR. CONCLUSIONS: AN680 is very effective in preventing DSS-induced UC in mice and may therefore have potential therapeutic application in humans. Addition of ChE inhibition and indirect activation of α7nAChR lessens the efficacy of AN917 in this model.

Ly6Chi Monocytes and Their Macrophage Descendants Regulate Neutrophil Function and Clearance in Acetaminophen-Induced Liver Injury.

Monocyte-derived macrophages (MoMF) play a pivotal role in the resolution of acetaminophen-induced liver injury (AILI). Timely termination of neutrophil activity and their clearance are essential for liver regeneration following injury. Here, we show that infiltrating Ly6Chi monocytes, their macrophage descendants, and neutrophils spatially and temporally overlap in the centrilobular necrotic areas during the necroinflammatory and resolution phases of AILI. At the necroinflammatory phase, inducible ablation of circulating Ly6Chi monocytes resulted in reduced numbers and fractions of reactive oxygen species (ROS)-producing neutrophils. In alignment with this, neutrophils sorted from monocyte-deficient livers exhibited reduced expression of NADPH oxidase 2. Moreover, human CD14+ monocytes stimulated with lipopolysaccharide or hepatocyte apoptotic bodies directly induced ROS production by cocultured neutrophils. RNA-seq-based transcriptome profiling of neutrophils from Ly6Chi monocyte-deficient versus normal livers revealed 449 genes that were differentially expressed with at least twofold change (p ≤ 0.05). In the absence of Ly6Chi monocytes, neutrophils displayed gene expression alterations associated with decreased innate immune activity and increased cell survival. At the early resolution phase, Ly6Chi monocytes differentiated into ephemeral Ly6Clo MoMF and their absence resulted in significant accumulation of late apoptotic neutrophils. Further gene expression analysis revealed the induced expression of a specific repertoire of bridging molecules and receptors involved with apoptotic cell clearance during the transition from Ly6Chi monocytes to MoMF. Collectively, our findings establish a phase-dependent task division between liver-infiltrating Ly6Chi monocytes and their MoMF descendants with the former regulating innate immune functions and cell survival of neutrophils and the later neutrophil clearance.