A conserved long noncoding RNA, GAPLINC, modulates the immune response during endotoxic shock Apple Cortez Vollmersa, Sergio Covarrubiasa, Daisy Kuanga, Aaron Shulkina, Justin Iwuagwua, Sol Katzmanb, Ran Songc, Kasthuribai Viswanathanc, Christopher Vollmersd, Edward Wakelandc, and Susan Carpentera,1 aDepartment of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064; bGenomics Institute, University of California, Santa Cruz, CA 95064; cDepartment of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9093; and dDepartment of Biomolecular Engineering, University of California, Santa Cruz, CA 95064 Edited by Ruslan Medzhitov, Yale University, New Haven, CT, and approved December 18, 2020 (received for review August 6, 2020) Recent studies have identified thousands of long noncoding RNAs function (4–7), we wanted to investigate the role of lncRNAs in (lncRNAs) in mammalian genomes that regulate gene expression macrophages. Macrophages are important innate immune cells in different biological processes. Although lncRNAs have been that can be derived from monocytes and are critical for pathogen identified in a variety of immune cells and implicated in immune recognition through the use of Toll-like receptors (TLRs). Upon response, the biological function and mechanism of the majority activation, TLRs initiate complex signaling pathways that acti- remain unexplored, especially in sepsis. Here, we identify a role for vate key transcription factors such as NF-κB, leading to the a lncRNA—gastric adenocarcinoma predictive long intergenic non- transcription of hundreds of immune response genes (8). coding RNA (GAPLINC)—previously characterized for its role in To identify lncRNAs involved in macrophage differentiation cancer, now in the context of innate immunity, macrophages, and function, we conducted RNA sequencing (RNA-Seq) in and LPS-induced endotoxic shock. Transcriptome analysis of mac- both human primary monocyte-derived macrophages (MDMs) rophages from humans and mice reveals that GAPLINC is a con- and the monocytic cell line THP-1s (Fig. 1A and SI Appendix, served lncRNA that is highly expressed following macrophage Fig. S1). We identified gastric adenocarcinoma predictive long differentiation. Upon inflammatory activation, GAPLINC is rapidly intergenic noncoding RNA (GAPLINC) as the most up- down-regulated. Macrophages depleted of GAPLINC display en- regulated lncRNA during monocyte to macrophage differentia- hanced expression of inflammatory genes at baseline, while over- tion (Fig. 1B). GAPLINC levels were detectable by day 1 and expression of GAPLINC suppresses this response. Consistent with increased to ∼300 copies per cell (Fig. 1C). Using RNA-profiling IMMUNOLOGY AND INFLAMMATION GAPLINC-depleted cells, Gaplinc knockout mice display enhanced technology (nCounter, Nanostring), we validated GAPLINC as basal levels of inflammatory genes and show resistance to LPS- one of the top 10 mRNAs expressed in differentiated primary induced endotoxic shock. Mechanistically, survival is linked to in- MDMs and THP-1s (Fig. 1D). We also confirmed that creased levels of nuclear NF-κBinGaplinc knockout mice that GAPLINC is highly expressed in MDMs but not expressed in the drives basal expression of target genes typically only activated closely related cell type monocyte-derived dendritic cells following inflammatory stimulation. We show that this activation (MDDCs) (Fig. 1E), suggesting that expression of GAPLINC is of immune response genes prior to LPS challenge leads to de- cell-type specific. By performing a cell fractionation experiment creased blood clot formation, which protects Gaplinc knockout and measuring GAPLINC levels in the cytoplasmic and nuclear mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative Significance regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock. Inflammation has largely been studied in the context of protein-coding genes. Recent studies have uncovered lncRNAs long noncoding RNA | inflammation | GAPLINC | innate immunity | sepsis as important regulators of immunity. The functional charac- terization of these genes remains an active area of research. In epsis is a life-threatening illness caused by an overreaction of this study, we identify GAPLINC as a functionally conserved Sthe body to the presence of infection, which can rapidly lead lncRNA between human and mouse. GAPLINC depletion results to multiorgan failure and death. The immune system is essential in enhanced expression of immune response genes that are in providing protection against infection; however, uncontrolled direct NF-κB targets. Astoundingly, we observe that Gaplinc activation can have serious consequences for the host. According knockout mice show resistance to LPS-induced endotoxic shock to the Centers for Disease Control and Prevention, one in three and find that basal expression of inflammatory genes prevents patients who die in a hospital have sepsis (1), and yet we still do clot formation to protect against multiorgan failure and death. not understand the underlying molecular mechanisms that lead These findings have implications in the treatment of sepsis, in to fatality. Clinical options for the treatment of sepsis are limited which new therapies targeting lncRNAs can contribute valu- to the delivery of fluid, antibiotics, and supportive care and have able information in understanding inflammation and improv- remained largely unchanged for decades. Though early diagno- ing patient outcome. ses and rapid treatment have improved sepsis outcomes (2), there is a critical need to develop new therapies. Although gene Author contributions: A.C.V., S. Covarrubias, and S. Carpenter designed research; A.C.V., S. Covarrubias, D.K., J.I., R.S., K.V., and S. Carpenter performed research; C.V. contributed expression studies have been performed to examine potential new reagents/analytic tools; A.C.V., S. Covarrubias, A.S., S.K., E.W., and S. Carpenter an- therapeutic targets for sepsis, these targets remain largely alyzed data; and A.C.V. and S. Carpenter wrote the paper. uncharacterized (3). We have identified a long noncoding RNA The authors declare no competing interest. (lncRNA) with roles in controlling the immune response and This article is a PNAS Direct Submission. endotoxic shock that provides avenues for novel drug develop- This open access article is distributed under Creative Commons Attribution-NonCommercial- ment to target sepsis. NoDerivatives License 4.0 (CC BY-NC-ND). 1To whom correspondence may be addressed. Email: [email protected]. Results This article contains supporting information online at https://www.pnas.org/lookup/suppl/ As lncRNA expression can regulate the immune response by doi:10.1073/pnas.2016648118/-/DCSupplemental. affecting immune cell differentiation and their respective Published February 10, 2021. PNAS 2021 Vol. 118 No. 7 e2016648118 https://doi.org/10.1073/pnas.2016648118 | 1of9 Downloaded by guest on September 29, 2021 A PBMCs BC10-20 RNA-seq (mac/mono) 400 GAPLINC -15 +MCSF 10 300 isolate macrophages monocytes 10-10 200 THP-1 +PMA p-value macrophages 10-5 100 cells Copies per cell 100 0 -10 -5 0 5 10 01234567 log2 Fold-Change Days relative D GMP min max E dendritic cell macrophage Mono1 Mono2 Mac1 Mac2 Mac3 Mac4 THP-1_1 THP-1_2 THP-1_3 THP-1_PMA1 THP-1_PMA2 THP-1_PMA3 Gene Name COL4A2-AS2 GAPLINC RefSeq Genes GAPLINC L3MBTL4-AS1 RP11-134G8 60 RP11-184M15 Monocytes RP11-20G13 1 60 RP11-20G13 Dendritic Cells RP11-343J3 1 60 RP11-519G16 read density Macrophages TMEM26-AS1 1 G F 8 100 Cyto input RNA 6 75 Nuc 60S-80S polysomes 50 4 Percent 25 2 0 0 fold (normalized to input RNA) C CD14 CD14 NEAT1 NEAT1 GAPLINC GAPLIN Fig. 1. Identification and characterization of macrophage-specific lncRNA GAPLINC. (A) A schematic for macrophage differentiation in vitro using primary human cells or immortalized THP-1 cells. Isolated monocytes from human PBMCs are differentiated into macrophages using recombinant macrophage colony- stimulating factor. THP-1 cells are differentiated into macrophages by treatment with PMA (100 nM). (B) RNA-Seq analysis on macrophages differentiated from monocytes isolated from human PBMCs (n = 4 donors). Results are represented in a volcano plot. GAPLINC (shown in red) is the most up-regulated lncRNA (>1,000-fold). (C) RNA-Seq analysis of GAPLINC expression during monocyte to macrophage differentiation for the indicated time points. GAPLINC expression is represented as copies per cell (FPKM). (D) Heat map represents gene expression from a custom Nanostring panel, which shows the top 10 differentially expressed lncRNA comparing monocytes to macrophages in primary human cells and THP-1 cells. Data from Nanostring were performed in biological duplicates. (E) A schematic for granulocyte-monocyte progenitor cells that give rise to two distinct populations: 1) MDDCs and 2) MDMs. UCSC genome browser track displays RNA-Seq reads from monocytes, macrophages, and dendritic cells at the GAPLINC locus. (F) qPCR analysis of RNAs purified from nuclear (white) and cytoplasmic (gray) fractions in MDMs. (G) qPCR analysis of RNAs isolated from different polysome fractions of MDM lysates (10 to 50% sucrose gradient, SW41, 40 K rpm, for ∼1.5 h). compartments of macrophages using qPCR, we found and guanylate-binding proteins (GBPs) (GBP3 and GBP5) GAPLINC is predominantly localized in the cytosol when com- (Fig. 2C and SI Appendix, Fig.