Background Microglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity. Methods Microglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity. Results We report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity. Conclusions Our data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.
To adequately reduce new HIV infections, development of highly effective pre-exposure prophylaxis (PrEP) against HIV infection in women is necessary. Cellulose acetate phthalate (CAP) is a pH sensitive polymer with HIV-1 entry inhibitory properties. Dolutegravir (DTG) is an integrase strand transfer inhibitor with potent antiretroviral activity. DTG delivered in combination with CAP may significantly improve current PrEP against HIV. In the present study the development of DTG-loaded CAP nanoparticles incorporated in thermosensitive (TMS) gel at vaginal pH 4.2 and seminal fluid pH 7.4 is presented as proof-of-concept for improved PrEP. Water–oil–in–water homogenization was used to fabricate DTG-loaded CAP nanoparticles (DTG–CAP–NPs). Size, polydispersity, and morphological analyses illustrate that DTG–CAP–NPs were smooth and spherical, ≤200 nm in size, and monodispersed with a polydispersity index PDI ≤ 0.2. The drug encapsulation (EE%) and release profile of DTG–CAP–NPs was determined by HPLC analysis. The EE% of DTG in DTG–CAP–NPs was evaluated to be ∼70%. The thermal sensitivity of the TMS gel was optimized and the pH dependency was evaluated by rheological analysis. DTG release studies in TMS gel revealed that DTG–CAP–NPs were stable in TMS gel at pH 4.2 while DTG–CAP–NPs in TMS gel at pH 7.4 rapidly release DTG (≥80% release within 1 h). Cytotoxicity studies using vaginal cell lines revealed that DTG–CAP–NPs were relatively non-cytotoxic at concentration <1 μg/mL. Confocal microscopic studies illustrate that ≥98% cells retained DTG–CAP–NPs intracellularly over seven days. Antiretroviral drug loaded nanocellulose fabrications in TMS gel delivered intravaginally may enhance both microbicidal and antiretroviral drug efficacy and may present a novel option for female PrEP against HIV.
The cells of the intestinal epithelium establish the frontline for host defense against pathogens in the gastrointestinal tract and play a vital role in the initiation of the immune response. Increasing evidence supports the role of long non-coding RNAs (lncRNAs) as critical regulators of diverse cellular processes, however, their role in antimicrobial host defense is incompletely understood. In this study, we provide evidence that the lncRNA Nostrill is upregulated in the intestinal epithelium following infection by Cryptosporidium parvum, a globally prevalent apicomplexan parasite that causes significant diarrheal disease and an important opportunistic pathogen in the immunocompromised and AIDS patients. Induction of Nostrill in infected intestinal epithelial cells was triggered by NF-κB signaling and was observed to enhance epithelial defense by decreasing parasitic infection burden. Nostrill participates in the transcriptional regulation of C. parvum-induced Irf7 expression through interactions with NF-κB p65, and induction of Nostrill promotes epigenetic histone modifications and occupancy of RNA polymerase II at the Irf7 promoter. Our data suggest that the induction of Nostrill promotes antiparasitic defense against C. parvum and enhances intestinal epithelial antimicrobial defense through contributions to transcriptional regulation of immune-related genes, such as Irf7.
Background Microglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by environmental microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNA (lncRNA) are emerging as important tissue-specific regulators directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Methods Microglial gene expression array analyses and qRT-PCR was used to identify a novel intergenic long-noncoding RNA that was upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin RNA immunoprecipitation assays, and qRT-PCR were used to study the function of this long-noncoding RNA in microglia. In vitro cytotoxicity assays were used to examine the effects of silencing the novel long-noncoding RNA in LPS-stimulated microglia on neurotoxicity. Results We report here that the previously uncharacterized intergenic lncRNA we termed Nostrill is induced by LPS stimulation in both BV2 cells and primary murine microglia, as well as in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and nitric oxide production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrate that silencing of Nostrill in microglia reduced LPS-stimulated microglia neurotoxicity. Conclusions Our data indicate a new regulatory role of NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide in microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.
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