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Bipolar disorder (BD) is psychiatric disorder of not fully acknowledged pathophysiology. Studies show the involvement of innate-immune system activation and inflammation in BD course and treatment efficiency. Microglia are crucial players in the inflammatory response possibly responsible for BD innate-immune activity. Lithium is a mood stabilizer used in treatment for 75 years. Immunomodulation was previously described as one of the potential modes of its action. We hypothesized that lithium might modulate the microglia response to innate-immune-associated cytokines (10 ng/mL TNF-α, 50 ng/mL IL-1β, 20 ng/mL IFN-γ). We aimed to investigate whether lithium treatment and pretreatment of microglia modify the expression of genes associated with NLRP3 inflammasome. We also aimed to verify lithium treatment effect on caspase activity and extracellular IL-1β concentration. For the first time, our study used human microglial cell line - HMC3, the cytokine stimuli and lithium in concentration corresponding to that in the brains of patients. To analyze lithium mode of action, we analyzed the short- and long-term treatment and pretreatment. To assess the influence on microglia responding to innate-immune cytokines, we analyzed the expression of genes involved in innate-immune and inflammasome (TSPO, TLR4, NFKB1, CASP1, CASP4, NLRP3, IL-1β, IL-6), caspase activity, extracellular IL-1β concentration, phospho-GSK-3β(Ser9) expression and lactate concentration. We found that lithium treatment significantly reduced NLRP3 inflammasome-related genes expression. We observed that lithium treatment reduces inflammasome activity, which may attenuate the inflammatory state. Interestingly, the lithium pretreatment resulted in significantly elevated inflammasome activity, suggesting that lithium does not impair the immune response to additional stimuli., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Asthma is a major non-communicable disease whose pathogenesis is still not fully elucidated. One of the asthma research models is precision-cut lung slices (PCLSs), and among the therapeutic options, miRNA molecules are of great interest. The aim of our study was to investigate whether inhibition of miR-223-3p and miR328a-3p affects the inflammatory response in PCLSs derived from a rat with HDM-induced allergic inflammation and a control rat. We generated rat PCLSs and transfected them with miR-223-3p and miR-328a-3p inhibitors. RNA was isolated from PCLSs and analyzed by qPCR. We also examined the proteins in the culture medium using the Magnetic Luminex Assay. The comparison between miRNA-transfected PCLSs and non-transfected controls showed significant differences in the expression of several genes associated with allergic inflammation, including Il-33 , Ccl5 , Prg2 and Tslp , in both the rat with allergic inflammation and the control rat. In the culture medium, we found no significant differences in protein levels between rat with allergic inflammation and the control. Our study highlighted some important issues: the need to extend the model by including more biological replicates, the need to standardize culture conditions, and the need to consider co-transfection with several miRNA inhibitors when modifying miRNAs expression in the PCLS model.

Depression is a common disease that affects 3.8% of the global population. Despite various antidepressant treatments, one-third of patients do not respond to antidepressants, therefore augmentation with mood stabilizers such as lithium may be required in this group. One of the suggested pathomechanisms of depression is the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and recent reports showed that microRNAs (miRNA) can impact its activity by epigenetic regulation. We aimed to explore the miRNA expression profile in the depression model and its changes upon short-term and chronic lithium treatment in the rat brain (pituitary, hypothalamus, and hippocampus). We used a chronic mild stress rat model of depression and short- and long-term lithium treatment. The behavior was assessed by an open-field test. The miRNA expression profile in the pituitary was estimated by sequencing and validated in the hypothalamus and hippocampus with qPCR. We found several miRNAs in the pituitary that were significantly altered between CMS-exposed and control rats as well as after short- and long-term lithium treatment. MicroRNAs chosen for validation in the hypothalamus and hippocampus (rno-miR-146a-5p, rno-miR-127-3p) showed no significant changes in expression. We performed in silico analysis and estimated potential pathways involved in lithium action for miRNAs differentially expressed in the pituitary at different time points. Specific microRNA subsets showed altered expression in the pituitary in depression model upon short- and long-term lithium treatment. We identified that biological pathways of target genes for these altered miRNAs differ, with the Foxo pathway potentially involved in disease development., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Precision-cut lung slices (PCLS) are ex vivo models with preserved lung cell populations and maintained tissue architecture. PCLS are, therefore, a powerful tool in respiratory research to study molecular mechanisms that closely reflect whole tissue biology. High-quality RNA and protein extraction from PCLS is, however, challenging as agarose significantly interferes with the yield and purity of extracted material. The present study aimed to optimize QIAzol-based isolation protocol for high-yield and quality RNA, miRNA, and protein extraction from PCLS., Materials and Methods: PCLS were prepared from 10 to 15-week-old Wistar rats and cultured for 7 days in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 0.1% FBS, penicillin, and streptomycin. LDH release to PCLS culture media was measured to determine cellular cytotoxicity. To select the optimal miRNA/RNA isolation protocol, we tested two different times (10 min, 2 h) and temperatures (room temperature, 4 °C, and -20 °C) of precipitation with isopropanol. Finally, we also assessed isolation with GHCL (guanidinium hydrochloride) extraction buffer. To select the optimal protein isolation protocol, we tested protein precipitation for 10 min at room temperatures (21 ± 1 °C) with 1.5 volumes of isopropanol and 3 volumes of acetone per 1 volume of phenol-ethanol supernatant. Additionally, we also tested protein precipitation for 3 h at -20 °C with 3, 5, and 7 acetone volumes per 1 volume of phenol-ethanol supernatant. We also validated protein precipitation with back extraction buffer instead of 100% ethanol. To measure the general efficiency of the optimized QIAZ-4 protocol, we used native rat lungs. PCLS for the ex vivo model of allergic inflammation were treated with IL-13 at a concentration of 80 ng/ml., Results: Standard QIAzol isolation protocol provided RNA, miRNA, and protein with low yield and poor quality. We found that 2-h isopropanol precipitation at 4 °C with a high concentration of salts significantly increased the yield and quality of extracted RNA and miRNA and provided acceptable qPCR efficiency (between 90 and 110%). Surprisingly, 2-h isopropanol precipitation at -20 °C significantly increased qPCR efficiency above the acceptable range (average efficiency: 120.4%). As for protein extraction, we found that 3-h acetone precipitation at -20 °C provided the highest yield with linear protein detection on Westen Blot. Optimized QIAZ-4 provided significantly higher miRNA and RNA yield compared to standard QIAzol protocols. We also found a significantly increased expression of Eotaxin-1 in PCLS treated with IL-13 as compared to the untreated controls., Conclusions: In our study, we described a simple QIAzol-based method for the simultaneous isolation of RNA, miRNA, and protein from PCLS., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable (In accordance with local requirements, ethical approval was not required as animals were subjected to tissue sampling but not to experimental procedures). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Precision cut lung slices (PCLS) are complex three-dimensional (3-D) lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules [e.g., microRNA (miRNA)]. The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. PCLS of 4 mm diameter were generated using agarose-filled rodent lungs and a vibratome. TYE665-labeled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualized using live high-content fluorescence microscopy, followed by higher-resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2, we established that cellular membranes in PCLS are permeable in the initial 24 h of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 h after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared with other LNPs, with detection possible as low as 25 nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs. NEW & NOTEWORTHY RNA-based therapeutics hold significant promise for disease treatment; however, limited research exists on miRNA transfection specifically within PCLS. miRNA transfection has thus far required custom functionalization for stabilization and internalization. We aimed to optimize a transfection protocol for rapid screening approaches of miRNA sequences. We show that transfecting miRNA in PCLS is possible using lipid nanoparticles. In addition, we show that 25 nM of TYE665-miRNA is sufficient for detection in a high-content imaging system.

Background: Asthma is the most frequent chronic disease in children. One of the most replicated genetic findings in childhood asthma is the ORMDL3 gene confirmed in several GWA studies in several pediatric populations., Objectives: The purpose of this study was to analyze ORMDL3 variants and expression in childhood asthma in the Polish population., Methods: In the study we included 416 subject, 223 asthmatic children and 193 healthy control subjects. The analysis of two SNPs (rs3744246 and rs8076131) was performed using genotyping with TaqMan probes. The methylation of the ORMDL3 promoter was examined with Methylation Sensitive HRM (MS-HRM), covering 9 CpG sites. The expression of ORMDL3 was analyzed in PBMCs from pediatric patients diagnosed with allergic asthma and primary human bronchial epithelial cells derived from healthy subjects treated with IL-13, IL-4, or co-treatment with both cytokines to model allergic airway inflammation., Results: We found that ORMDL3 expression was increased in allergic asthma both in PBMCs from asthmatic patients as well as in human bronchial epithelial cells stimulated with the current cytokines. We did not observe significant differences between cases and controls either in the genotype distribution of analyzed SNPs (rs3744246 and rs8076131) nor in the level of promoter methylation., Conclusions: Increased ORMDL3 expression is associated with pediatric allergic asthma and upregulated in the airways upon Th2-cytokines stimulation, but further functional studies are required to fully understand its role in this disease.

Children with cystic fibrosis (CF) suffer from chronic inflammation and recurrent pulmonary exacerbations (PEs). We aimed to test whether a specific miRNA could be associated with the occurrence of PE. We sequenced extracellular vesicle (EV)-derived miRNA in sputum ( n = 20), exhaled breath condensate (EBC) ( n = 11), and serum ( n = 8) samples from pediatric patients during PE and the stable stage of CF. Four miRNAs: let-7c, miR-16, miR-25-3p and miR-146a, have been selected for validation in a larger group with reverse transcription quantitative real-time PCR (RT-qPCR) in sputum and serum, or droplet digital PCR (ddPCR) in EBC. Next-generation sequencing (NGS) differential expression analysis was done in Base Space, and the correlation between miRNAs expression and clinical data was calculated with Statistica. Functional annotation of selected miRNAs and their potential target genes was performed with miRDip and DAVID software. There were no differences in miRNA expression between stable and exacerbation in sputum and in serum. Validation of four selected miRNAs showed significant downregulation of miR-146a in serum. A panel of all four miRNAs (peripherally) was the best predictive model of exacerbation ( p < 0.001, AUC = 0.96). Expression of airway miR-25-3p improved the diagnostic value of FEV1% pred and FVC% pred, while peripheral miR-146a improved the predictive model of C-reactive protein and neutrophilia. In silico analysis revealed a potential role for selected miRNAs in regulating processes associated with inflammation and tissue remodeling. We demonstrated that EVs contained in peripheral blood as well as local biomaterials can act as carriers for miRNAs with the diagnostic potential of predicting exacerbation in pediatric CF., (© 2023 IOP Publishing Ltd.)

Introduction: Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described. The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation., Materials and Methods: As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the BaseSpace tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223-3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13., Results: We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223-3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223-3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes., Conclusions: These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223-3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223-3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared with animal models. Several experimental readouts have been established for use with PCLS, but obtaining high-yield and -quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for nonbiased and high-throughput analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high-quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis. We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.

Lithium has been the most important mood stabilizer used for the treatment of bipolar disorder and prophylaxis of manic and depressive episodes. Despite long use in clinical practice, the exact molecular mechanisms of lithium are still not well identified. Previous experimental studies produced inconsistent results due to different duration of lithium treatment and using animals without manic-like or depressive-like symptoms. Therefore, we aimed to analyze the gene expression profile in three brain regions (amygdala, frontal cortex and hippocampus) in the rat model of mania and depression during chronic lithium administration (2 and 4 weeks). Behavioral changes were verified by the forced swim test, open field test and elevated maze test. After the experiment, nucleic acid was extracted from the frontal cortex, hippocampus and amygdala. Gene expression profile was done using SurePrint G3 Rat Gene Expression whole transcriptome microarrays. Data were analyzed using Gene Spring 14.9 software. We found that chronic lithium treatment significantly influenced gene expression profile in both mania and depression models. In manic rats, chronic lithium treatment significantly influenced the expression of the genes enriched in olfactory and taste transduction pathway and long non-coding RNAs in all three brain regions. We report here for the first time that genes regulating olfactory and taste receptor pathways and long non-coding RNAs may be targeted by chronic lithium treatment in the animal model of mania.

Adipose tissue is a major source of circulating exosomal microRNAs (miRNAs) that are modulators of the immune response in various types of tissues and organs, including airways. Still, no evidence exists if allergic airway inflammation may affect fat tissue inflammation via alterations in the miRNA expression profile. Therefore, we investigated the miRNA expression profile in the adipose tissue upon induced allergic inflammation in the airways in the rat. Brown Norway rats were chronically sensitized to house dust mite extract for seven weeks. Body composition was performed using MiniSpec Plus. The eosinophil count and the total IgE level were determined to confirm the induction of allergic inflammation. MiRNA expression profiling was done using the next-generation sequencing with validation by qPCR. We found that allergic airway inflammation significantly increased fat in adipose tissue, glucose concentration, and the gene expression of adipose tissue-derived proinflammatory peptides (leptin, TNFα). In miRNA-seq analysis, we showed significant differences in the expression of 36 mature miRNAs, three precursors, and two miRNA families in adipose tissue of allergic rats. Two miRNAs-miRNA-151-5p and miRNA-423-3p-showed significantly increased expression in qPCR in adipose tissue and lungs of sensitized animals. Allergic airway inflammation affects fat tissue and alters miRNA expression profile in adipose tissue in the rat.

MicroRNAs are small non-coding RNAs that regulate immune response and inflammation. We assumed that miRNAs may be involved in the immune response during cystic fibrosis pulmonary exacerbations (CFPE) and that altered expression profile in the airways and blood may underlie clinical outcomes in CF pediatric patients., Methods: We included 30 pediatric patients diagnosed with cystic fibrosis. The biologic material (blood, sputum, exhaled breath condensate) was collected during pulmonary exacerbation and in stable condition. The miRNA expression profile from blood and sputum ( n = 6) was done using the next-generation sequencing. For validation, selected four miRNAs were analyzed by qPCR in exosomes from sputum supernatant and exhaled breath condensate ( n = 24). NGS analysis was done in Base Space, correlations of gene expression with clinical data were done in Statistica., Results: The miRNA profiling showed that four miRNAs (miR-223, miR-451a, miR-27b-3p, miR-486-5p) were significantly altered during pulmonary exacerbation in CF patients in sputum but did not differ significantly in blood. MiRNA differently expressed in exhaled breath condensate (EBC) and sputum showed correlation with clinical parameters in CFPE., Conclusion: MiRNA expression profile changes in the airways during pulmonary exacerbation in CF pediatric patients. We suggest that miRNA alterations during CFPE are restricted to the airways and strongly correlate with clinical outcome.

Methods: In the study, we included 86 children diagnosed with atopic asthma ( n = 25), allergic rhinitis ( n = 20), and atopic dermatitis ( n = 20) and healthy control subjects ( n = 21) of Caucasian origin from the Polish population. The blood leukocyte expression of 31 genes involved in neuroinflammatory response (neurotrophins, their receptors, neuropeptides, and histamine signaling pathway) was analysed using TaqMan low-density arrays. The relative expression of selected proteins from plasma was done using TaqMan Protein Assays. Statistical analysis was done using Statistica., Results: Blood expression of 31 genes related to neuroimmune interactions showed significant increase in both allergic diseases, allergic rhinitis and atopic dermatitis, in comparison to the control group. We found 12 genes significantly increased in allergic rhinitis and 9 genes in which the expression was elevated in atopic dermatitis. Moreover, 9 genes with changed expression in atopic dermatitis overlapped with those in allergic rhinitis. Atopic asthma showed 5 genes with altered expression. The peripheral expression of neuroinflammatory genes in the human study was verified in target tissues (nasal epithelium and skin) in a rat model of allergic inflammation., Conclusions: A common pattern of neuroinflammatory gene expression between allergic rhinitis and atopic dermatitis may reflect similar changes in sensory nerve function during chronic allergic inflammation., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Paulina Sobkowiak et al.)

Purpose: Airway epithelium acts as a protective barrier against the particles from the inhaled air. Damage to the epithelium may result in loss of the barrier function. Epithelial repair in response to injury requires complex mechanisms, such as microRNA, small noncoding molecules, to regulate the processes involved in wound repair. We aimed to establish if the microRNA gene expression profile is altered during the airway epithelial repair in differentiated cells., Methods: miRNA gene expression profile during the wound closure of differentiated normal human bronchial epithelium (NHBE) from one donor was analysed using quantitative real-time PCR. We have analysed the expression of 754 genes at five time points during a 48-hour period of epithelium repair using TaqMan Low Density Array., Results: We found out that 233 miRNA genes were expressed in normal human bronchial epithelium. Twenty miRNAs were differentially expressed during the wound repair process, but only one (miR-455-3p) showed significance after FDR adjustment ( p = 0.02). Using STEM, we have identified two clusters of several miRNA genes with similar expression profile. Pathway enrichment analysis showed several significant signaling pathways altered during repair, mainly involved in cell cycle regulation, proliferation, migration, adhesion, and transcription regulation., Conclusions: miRNA expression profile is altered during airway epithelial repair of differentiated cells from one donor in response to mechanical injury in vitro , suggesting their potential role in wound repair.

Background and Aim: Interleukin-1 is a pro-inflammatory cytokine found in two forms (α and β). The α form is mainly cell-bound, whereas IL-1β is primarily secreted by macrophages in response to immune system stimulation. We hypothesized that polymorphic variants of interleukin 1 genes may play a role in childhood asthma risk. The aim of this study was to investigate if IL-1α and β polymorphism is associated with asthma in a pediatric population and if the genotype affects its serum level., Methods: The studied population included 310 children aged 6-18 years old (152 with asthma and 158 healthy children). Genotypes were determined with real-time PCR method using TaqMan Genotyping Assays. Serum level was measured with ELISA Set. Statistical analysis was done in Statistica v.12.0. Linkage disequilibrium and haplotype analysis was done in Haploview v. 4.2., Results: We found that three IL-1β polymorphisms rs1143634, rs1143633, and rs1143643 were associated with allergic asthma risk (P = 0.034; OR = 1.523; P = 0.024, OR = 1.477; 0.044, OR = 1.420, respectively). We also found a strong linkage disequilibrium between these polymorphisms and CAC haplotype was associated significantly with asthma risk (P = 0.023). For IL1α, we did not observe association with asthma. We then analyzed if IL-1β expression was altered in serum and we found that asthmatic children showed significantly higher IL-1β levels than healthy controls (P = 0.047). No association with asthma was observed for IL-1 α variants., Conclusions: This study indicates that IL-1β gene polymorphism may affect allergic asthma risk in children., (© 2017 Wiley Periodicals, Inc.)

Non-coding RNAs (ncRNAs) are involved in the regulation of numerous biological processes and pathways and therefore have been extensively studied in human diseases. Previous reports have shown that non-coding RNAs play a crucial role in the pathogenesis and aberrant regulation of respiratory diseases. The altered expression of microRNAs (miRNAs) and long noncoding RNAs in blood and also locally in sputum or exhaled breath condensate influences lung function, immune response, and disease phenotype and may be used for the development of biomarkers specific for airway disease. In this review, we provide an overview of the recent works studying the non-coding RNAs in airway diseases, with a particular focus on chronic respiratory diseases of childhood. We have chosen the most common chronic respiratory condition-asthma- and the most severe, chronic disease of the airways-cystic fibrosis. Study of the altered expression of non-coding RNAs in these diseases may be key to better understanding their pathogenesis and improving diagnosis, while also holding promise for the development of therapeutic strategies using the regulatory potential of non-coding RNAs., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.