Biochemical and transcriptomic evaluation of a 3D lung organoid platform for pre-clinical testing of active substances targeting senescence

Chronic lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis are incurable. Epithelial senescence, a state of dysfunctional cell cycle arrest, contributes to the progression of such diseases. Therefore, lung epithelial cells are a valuable target for therapeutic intervention. Here, we present a 3D airway lung organoid platform for the preclinical testing of active substances with regard to senescence, toxicity, and inflammation under standardized conditions in a 96 well format. Senescence was induced with doxorubicin and measured by activity of senescence associated galactosidase. Pharmaceutical compounds such as quercetin antagonized doxorubicininduced senescence without compromising organoid integrity. Using single cell sequencing, we identified a subset of cells expressing senescence markers which was decreased by quercetin. Doxorubicin induced the expression of detoxification factors specifically in goblet cells independent of quercetin. In conclusion, our platform enables for the analysis of senescence-related processes and will allow the pre-selection of a wide range of compounds (e.g. natural products) in preclinical studies, thus reducing the need for animal testing

Methylglyoxal Mediates Adipocyte Proliferation by Increasing Phosphorylation of Akt1

Methylglyoxal (MG) is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5–20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE) breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity

Allergic lung inflammation alters neither susceptibility to Streptococcus pneumoniae infection nor inducibility of innate resistance in mice

<p>Abstract</p> <p>Background</p> <p>Protective host responses to respiratory pathogens are typically characterized by inflammation. However, lung inflammation is not always protective and it may even become deleterious to the host. We have recently reported substantial protection against <it>Streptococcus pneumoniae </it>(pneumococcal) pneumonia by induction of a robust inflammatory innate immune response to an inhaled bacterial lysate. Conversely, the allergic inflammation associated with asthma has been proposed to promote susceptibility to pneumococcal disease. This study sought to determine whether preexisting allergic lung inflammation influences the progression of pneumococcal pneumonia or reduces the inducibilty of protective innate immunity against bacteria.</p> <p>Methods</p> <p>To compare the effect of different inflammatory and secretory stimuli on defense against pneumonia, intraperitoneally ovalbumin-sensitized mice were challenged with inhaled pneumococci following exposure to various inhaled combinations of ovalbumin, ATP, and/or a bacterial lysate. Thus, allergic inflammation, mucin degranulation and/or stimulated innate resistance were induced prior to the infectious challenge. Pathogen killing was evaluated by assessing bacterial CFUs of lung homogenates immediately after infection, the inflammatory response to the different conditions was evaluated by measurement of cell counts of bronchoalveolar lavage fluid 18 hours after challenge, and mouse survival was assessed after seven days.</p> <p>Results</p> <p>We found no differences in survival of mice with and without allergic inflammation, nor did the induction of mucin degranulation alter survival. As we have found previously, mice treated with the bacterial lysate demonstrated substantially increased survival at seven days, and this was not altered by the presence of allergic inflammation or mucin degranulation. Allergic inflammation was associated with predominantly eosinophilic infiltration, whereas the lysate-induced response was primarily neutrophilic. The presence of allergic inflammation did not significantly alter the neutrophilic response to the lysate, and did not affect the induced bacterial killing within the lungs.</p> <p>Conclusion</p> <p>These results suggest that allergic airway inflammation neither promotes nor inhibits progression of pneumococcal lung infection in mice, nor does it influence the successful induction of stimulated innate resistance to bacteria.</p

Spontaneous Eosinophilic Nasal Inflammation in a Genetically-Mutant Mouse: Comparative Study with an Allergic Inflammation Model

Background: Eosinophilic inflammation is a hallmark of chronic rhinosinusitis with nasal polyps. To model this disease process experimentally, nasal sensitization of mice with ovalbumin or aspergillus has been described. Here, we describe a genetically mutant mouse that develops robust spontaneous nasal eosinophilic inflammation. These mice lack the enzyme SHP-1 that down-regulates the IL-4Ra/stat6 signaling pathway. We compared nasal inflammation and inflammatory mediators in SHP-1 deficient mice (mev) and an ovalbumin-induced nasal allergy model. Methods: A novel technique of trans-pharyngeal nasal lavage was developed to obtain samples of inflammatory cells from the nasal passages of allergic and mev mice. Total and differential cell counts were performed on cytospin preparations. Expression of tissue mRNA for IL-4, IL-13, and mouse beta-defensin-1 (MBD-1) was determined by quantitative PCR. Eotaxin in the lavage fluid was assessed by ELISA. Results: Allergic and mev mice had increased total cells and eosinophils compared with controls. Expression of IL-4 was similarly increased in both allergic and mev mice, but expression of IL-13 and eotaxin was significantly greater in the allergic mice than mev mice. Eotaxin was significantly up-regulated in both allergic rhinitis and mev mice. In both models of eosinophilic inflammation, down-regulation of the innate immune marker MBD-1 was observed. Conclusions: The mev mice display spontaneous chronic nasal eosinophilic inflammation with potential utility for chroni

miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449−/− mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449−/− mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449−/− mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449−/− cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis

Protection From Retinopathy and Other Complications in Patients With Type 1 Diabetes of Extreme Duration: The Joslin 50-Year Medalist Study

Objective: To assess complication prevalence and identify protective factors in patients with diabetes duration of $\geq$50 years. Characterization of a complication-free subgroup in this cohort would suggest that some individuals are protected from diabetes complications and allow identification of endogenous protective factors. Research Design and Methods: Cross-sectional, observational study of 351 U.S. residents who have survived with type 1 diabetes for $\geq$50 years (Medalists). Retinopathy, nephropathy, neuropathy, and cardiovascular disease were assessed in relation to HbA$_{1c}$, lipids, and advanced glycation end products (AGEs). Retrospective chart review provided longitudinal ophthalmic data for a subgroup. Results: A high proportion of Medalists remain free from proliferative diabetic retinopathy (PDR) (42.6%), nephropathy (86.9%), neuropathy (39.4%), or cardiovascular disease (51.5%). Current and longitudinal (the past 15 years) glycemic control were unrelated to complications. Subjects with high plasma carboxyethyl-lysine and pentosidine were 7.2-fold more likely to have any complication. Of Medalists without PDR, 96% with no retinopathy progression over the first 17 years of follow-up did not experience retinopathy worsening thereafter. Conclusions: The Medalist population is likely enriched for protective factors against complications. These factors might prove useful to the general population with diabetes if they can be used to induce protection against long-term complications. Specific AGE combinations were strongly associated with complications, indicating a link between AGE formation or processing with development of diabetic vasculopathy