Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.
ACRBP/sp32 is a binding protein specific for the precursor (pro-ACR) and intermediate forms of sperm serine protease ACR. In this study, we examined the expression pattern, localization, and possible role of mouse ACRBP in spermatogenic cells and epididymal sperm. Unlike other mammalian ACRBPs, two forms of Acrbp mRNA—wild-type Acrbp-W and variant Acrbp-V5 mRNAs—were generated by alternative splicing of Acrbp in the mouse. ACRBP-W was synthesized in pachytene spermatocytes and haploid spermatids and immediately processed into a mature protein, ACRBP-C, by removal of the N-terminal half. The intron 5-retaining splice variant mRNA produced a predominant form of ACRBP, ACRBP-V5, that was present in pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. ACRBP-W and ACRBP-V5 were both colocalized with pro-ACR in the acrosomal granules of early round spermatids, whereas the sperm acrosome contained only ACRBP-C. Glutathione S-transferase pull-down assays revealed that ACRBP-V5 and ACRBP-C possess a different domain capable of binding each of two segments in the C-terminal region of pro-ACR. Moreover, autoactivation of pro-ACR was remarkably accelerated by the presence of ACRBP-C. These results suggest that ACRBP-V5 and ACRBP-C may function in the transport/packaging of pro-ACR into acrosomal granules during spermiogenesis and in the promotion of ACR release from the acrosome during acrosomal exocytosis, respectively.
Abstract Background Alzheimer`s disease (AD) is a progressive neurodegenerative disease worldwide. Accumulation of amyloid-β (Aβ), neurofibrillary tangles and neuroinflammation play the important neuro-pathology in patients with AD. miRNA is multifunctional and involved in physiological and pathological processes. Recently, microRNAs have been linked to neurodegenerative diseases. However, it is little known whether miRNA dysregulation contributes to AD pathology progression such as Aβ processing, phagocytosis and neuroinflammation. Here, we identify miR485-3p as a novel modulator of AD pathology in 5XFAD mice. Methods To study the role of miR485-3p in AD, we used in control or miR485-3p antisense oligonucleotides (miR485-3p ASO) injected 5XFAD mouse model. Changes of Aβ processing and clearance and inflammation were analyzed by biochemical method in vitro and in vivo. Result This study suggests that miR485-3p, a novel miRNA targeting SIRT1 may contribute to pathogenesis in an AD mouse. We found SIRT1 is significantly reduced in the precentral gyrus of Alzheimer patient`s and in 5XFAD mice. To determine whether the inhibition of miRNA 485-3p would affect AD pathology, we studied the effect of the antisense oligo in the brain of 5XFAD mice through direct intracerebral ventricular injection with miR485-3p ASO. We demonstrated that miR485-3p ASO significantly reduced Aβ plaque and amyloid biosynthetic enzyme. Importantly, the attenuation of Aβ plaques through miR485-3p ASO was mediated through Aβ phagocytic activity of glial cells, by which it can directly target CD36. MiR485-3p ASO also decreased inflammatory responses. Collectively, these responses inhibited neuronal loss caused by Aβ lead to improvements of cognitive impairment. Conclusion Our data provide evidence for the molecular mechanisms which underlie the miR485-3p ASO responses in an AD mouse model. These results suggest that attenuating miRNA 485-3p levels might represent a novel therapeutic approach in AD.
Alzheimer’s disease (AD) is a form of dementia characterized by progressive memory decline and cognitive dysfunction. With only one FDA-approved therapy, effective treatment strategies for AD are urgently needed. In this study, we found that microRNA-485-3p (miR-485-3p) was overexpressed in the brain tissues, cerebrospinal fluid, and plasma of patients with AD, and its antisense oligonucleotide (ASO) reduced Aβ plaque accumulation, tau pathology development, neuroinflammation, and cognitive decline in a transgenic mouse model of AD. Mechanistically, miR-485-3p ASO enhanced Aβ clearance via CD36-mediated phagocytosis of Aβ in vitro and in vivo. Furthermore, miR-485-3p ASO administration reduced apoptosis, thereby effectively decreasing truncated tau levels. Moreover, miR-485-3p ASO treatment reduced secretion of proinflammatory cytokines, including IL-1β and TNF-α, and eventually relieved cognitive impairment. Collectively, our findings suggest that miR-485-3p is a useful biomarker of the inflammatory pathophysiology of AD and that miR-485-3p ASO represents a potential therapeutic candidate for managing AD pathology and cognitive decline.
The innate immune system plays key roles in controlling Alzheimer's disease (AD), while secreting cytokines to eliminate pathogens and regulating brain homeostasis. Recent research in the field of AD has shown that the innate immune-sensing ability of pattern recognition receptors on brain-resident macrophages, known as microglia, initiates neuroinflammation, Aβ accumulation, neuronal loss, and memory decline in patients with AD. Advancements in understanding the role of innate immunity in AD have laid a strong foundation to elucidate AD pathology and devise therapeutic strategies for AD in the future. In this review, we highlight the present understanding of innate immune responses, inflammasome activation, inflammatory cell death pathways, and cytokine secretion in AD. We also discuss how the AD pathology influences these biological processes.
Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered undruggable; however, manually designing optimal ASO sequences can be labor intensive and time consuming, which potentially limits their broader application. To address this challenge, we introduce a platform, the ASOptimizer, a deep-learning-based framework that efficiently designs ASOs at a low cost. This platform not only selects the most efficient mRNA target sites but also optimizes the chemical modifications for enhanced performance. Indoleamine 2,3-dioxygenase 1 (IDO1) promotes cancer survival by depleting tryptophan and producing kynurenine, leading to immunosuppression through the aryl-hydrocarbon receptor (Ahr) pathway within the tumor microenvironment. We used ASOptimizer to identify ASOs that target IDO1 mRNA as potential cancer therapeutics. Our methodology consists of two stages: sequence engineering and chemical engineering. During the sequence-engineering stage, we optimized and predicted ASO sequences that could target IDO1 mRNA efficiently. In the chemical-engineering stage, we further refined these ASOs to enhance their inhibitory activity while reducing their potential cytotoxicity. In conclusion, our research demonstrates the potential of ASOptimizer for identifying ASOs with improved efficacy and safety.
The innate immune system represents the first line of defense against influenza viruses, which cause severe inflammation of the respiratory tract and are responsible for more than 650,000 deaths annually worldwide. mRNA vaccines are promising alternatives to traditional vaccine approaches due to their safe dosing, low-cost manufacturing, rapid development capability, and high efficacy. In this review, we provide our current understanding of the innate immune response that uses pattern recognition receptors to detect and respond to mRNA vaccination. We also provide an overview of mRNA vaccines, and discuss the future directions and challenges in advancing this promising therapeutic approach.
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