Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)–derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases

Neutrophil SHIP1 and PTEN Deficiency and Response to Sterile Injury

Neutrophils are the primary effector cells of the innate immune response. These highly motile cells rapidly move to sites of infection and injury where they eradicate pathogens and/or remove and restructure damaged tissues. The recruitment and activity of these cells must be tightly regulated because they have the potential to induce severe tissue damage with their potent effector functions. In an in vitro model of sterile injury, where no pathogens are present, we have identified a potential non-formylated peptide released from necrotic cells that attracts neutrophils to a site of injury via the chemotaxis-inducing G-protein coupled formyl peptide receptor 1. This as-of-yet unidentified ligand was not sensitive to enzymatic deformylation. In competitive chemotaxis assays, this signal from disrupted cells acts as an “end-target” chemoattractant, causing neutrophils to ignore competing gradients of the endogenous chemokine interleukin-8, and move precisely to a site of injury. Additionally, we have investigated the role of two lipid phosphatases, SHIP1 and PTEN, in neutrophil behavior. These two phosphatases antagonize phosphatidylinositol-3 kinase signaling, which is one of the main signaling pathways involved in controlling neutrophil chemotaxis. Using a targetable loxP/Cre recombinase gene excision system in the mouse, we have, for the first time, simultaneously depleted both these phosphatases in neutrophils. In contrast to mice in which neutrophils lack either one or the other, and have little outwardly apparent abnormalities, the resulting double deficient animals had a severe phenotype. These animals exhibited hematopoietic disturbances characterized by an increased proliferative capacity in populations of immature neutrophils, with an enhanced recruitment of neutrophils and, in certain cases other cells such macrophages, to solid organs such as the liver, spleen, brain, lungs. They displayed pronounced hepatomegaly and splenomegaly, occasional neurological disturbances, organ dysfunction, and typically expired at less than four weeks of age. Analysis of neutrophil behavior in very young animals revealed decreased chemotactic function in vivo in the liver. In vitro analyses revealed chemotactic impairments towards certain cues, coupled with increased numbers of cells responding to single gradients of chemoattractants. These studies provide critical insight into the role of SHIP1 and PTEN in neutrophil function, development, and homeostasis

Method for Absolute Quantification of Short Chain Fatty Acids via Reverse Phase Chromatography Mass Spectrometry

Short chain fatty acids (SCFAs; including acetate, propionate, and butyrate) are an important class of biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules by liquid chromatography mass spectrometry (LC-MS) is challenging due to their relatively poor chromatographic properties and low mass. Herein, we introduce SQUAD, a quantitative strategy for analyzing SCFAs using an aniline-based derivatization approach in conjunction with reverse-phase LC-MS/MS analysis. We show that this approach, when coupled to quantification by stable isotope dilution (SID), enables absolute quantification of biologically relevant SCFAs in complex biological samples. We evaluate the limits of detection using this approach, analyze sources of error affecting quantification, and provide practical guidelines for using this strategy. Moreover, we illustrate the utility of this technique via two representative biological applications where SCFA analysis is common: 1) SCFA quantification in the caecal contents of germ free versus conventionally raised specific pathogen free mice and 2) in an analysis of in vitro microbial cultures. </p

Nucleoside Reverse Transcriptase Inhibitors Possess Intrinsic Anti-Inflammatory Activity

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)–derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases