Capili, A. | bioRxiv Science

bioRxiv2022

LincRNA-Cox2 regulates smoke-induced inflammation in murine macrophages

Cigarette smoke (CS) exposure is a risk factor for many chronic diseases including chronic obstructive pulmonary disease (COPD), however the mechanism by which smoke exposure can alter homeostasis and bring about chronic inflammation is poorly understood. Here, we showcase a novel role for smoke in regulating long noncoding RNAs (lncRNAs), showing that it activates lincRNA-Cox2, which we previously characterized as functional in inflammatory regulation. Exposing lincRNA-Cox2 murine models to smoke in vivo confirmed lincRNA-Cox2 as a regulator of inflammatory gene expression in response to smoke both systemically and within the lung. We also report that lincRNA-Cox2 negatively regulates genes in smoked bone marrow derived macrophages exposed to LPS stimulation. In addition to the effects on lncRNAs, we also report dysregulated transcription and splicing of inflammatory protein-coding genes in the bone marrow niche following CS exposure in vivo. Collectively, this work provides insights into how innate immune signaling from gene expression to splicing is altered following in vivo exposure to CS and highlights an important new role for lincRNA-Cox2 in regulating immune genes following smoke exposure.

immunology↗

bioRxiv2019

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

AbstractMacrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation and maintenance of homeostasis. Understanding the genes involved in all aspects of macrophage biology is essential to gaining new insights into immune system dysregulation during diseases that range from autoinflammatory to cancer. Here we utilize high throughput clustered regularly interspaced short palindromic repeats (CRISPR) screening to generate a resource that identifies genes required for macrophage viability and function. First, we employ a pooled based CRISPR/Cas nuclease active screening approach to identify essential genes required for macrophage viability by targeting genes within coding exons. In addition, we also target 3UTRs to gain insights into new cis-regulatory regions that control expression of these essential genes. Second, using our recently generated NF-{kappa}B reporter macrophage line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen to identify regulators of inflammation. We identify a number of novel positive and negative regulators of the NF-{kappa}B pathway as well as unraveling complexities of the TNF signaling cascade showing it can function in an autocrine manner to negatively regulate the pathway. Utilizing a single complex library design we are capable of interrogating various aspects of macrophage biology, generating a resource for future studies.\n\nAuthor SummaryExcess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are important mediators of this inflammatory response. Defining the genes involved in their viability and effector function is needed to completely understand these two important aspects of macrophage biology. Here we screened over 21,000 genes and generated a resource guide of genes required for macrophage viability as well as novel positive and negative regulators of NF-{kappa}B signaling.

immunology↗