Biology subjects

Carpenter, S.

Publications and source records attributed to Carpenter, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Restricted MHC-II trafficking in Mycobacterium tuberculosis-infected M2-like macrophages limits CD4+ T cell activation

Recognition of infected macrophages by CD4+ T cells is essential to immune protection against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). However, not all infected macrophage subsets successfully elicit T cell activation. We recently discovered that M2-like macrophages fail to efficiently activate memory CD4+ T cells when infected with Mtb, yet successfully elicit T cell activation when loaded with peptides, {gamma}-irradiated bacteria, or Mtb whole cell lysate. Since the mechanisms underlying CD4+ T cell evasion by infected M2 but not M1-like macrophages remain underexplored, we sought to determine the genes and pathways unique to Mtb infection of M2-like cells, including alveolar macrophages. RNA sequencing of human macrophages infected with virulent Mtb identified enrichment of IL-10 and type I interferon (IFN) signaling genes, including IL10RA and HERC5, respectively, in infected M2-like monocyte-derived and alveolar macrophages. However, genes involved in MHC-II trafficking, such as AP1M2, were higher in infected M1-like macrophages. In complementary experiments using fluorescence microscopy and flow cytometry, we observed impaired trafficking of newly synthesized MHC-II to the plasma membrane of Mtb-infected M2-like macrophages despite high total surface MHC-II levels. Neutralization of IL-10 or knockdown of HERC5 restored MHC-II trafficking to the cell surface among infected M2-like macrophages and significantly enhanced activation of memory CD4+ T cells in an MHC-II-dependent manner. These findings identify coordinated IL-10 and type I IFN signaling as key mechanisms that restrict MHC-II trafficking to the plasma membrane in Mtb-infected M2-like macrophages, thereby limiting antigen presentation and CD4+ T cell activation. We propose that host-directed therapies targeting these pathways in infected alveolar macrophages will facilitate T cell recognition for the prevention or treatment of active TB. Author SummaryRecognition of infected macrophages by CD4+ T cells is essential to immune protection against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). However, not all infected macrophage subsets successfully elicit T cell activation. We recently discovered that M2-like macrophages fail to efficiently activate memory CD4+ T cells when infected with Mtb, yet they successfully elicit T cell activation when treated with peptides, {gamma}-irradiated bacteria, or Mtb whole cell lysate. In this study, we identified genes and pathways uniquely upregulated in Mtb-infected M2-like macrophages that are linked to inefficient CD4+ T cell activation, including IL-10 signaling and type I interferon (IFN) pathways. These pathways were linked to reduced MHC-II trafficking to the plasma membrane in Mtb-infected M2-like macrophages. Neutralization of IL-10 or knockdown of HERC5 restored MHC-II trafficking and augmented memory CD4+ T cell activation. Our study demonstrates that IL-10 signaling and type I IFN pathways play detrimental roles in macrophages during Mtb infection, impairing MHC-II trafficking and CD4+ T cell activation. Since lung-resident alveolar macrophages express a dominant M2-like phenotype, these findings suggest that targeting IL-10 and type I IFN signaling may offer a strategy to enhance CD4+ T cell-mediated immunity and improve TB outcomes.

immunology↗

bioRxiv2026

Integrative multiomics analysis and CRISPR screening identify functional noncanonical translation loci in the mouse immune system.

Ribosome profiling has revealed thousands of noncanonical translation events across mammalian genomes, yet functional characterization has overwhelmingly focused on proliferative fitness in cancer cell lines. Here, we present a comprehensive survey of noncanonical translation in the mouse immune system and its functional consequences in macrophages. By performing a unified Ribo-seq meta-analysis across 20 public mouse leukocyte datasets - spanning macrophages, dendritic cells, neutrophils, B cells, and T cells - we define a compendium of 22,276 noncanonical coding sequences (CDSs), including upstream ORFs (uORFs), downstream ORFs, and ORFs on noncoding RNAs and pseudogenes (ncORFs). Proteogenomic integration with reanalyzed mass spectrometry data prioritizes a high-confidence subset with detectable protein products, including pseudogene-encoded and lncRNA-encoded zinc finger proteins. To move beyond cataloging, we carried out two orthogonal CRISPR screens in immortalized bone marrow-derived macrophages: a fitness screen identifying noncanonical CDSs required for macrophage viability, and a TLR1/TLR2-NF{kappa}B reporter screen uncovering CDSs that modulate innate immune signaling. These screens nominate uORFs, several conserved between mouse and human, that exert phenotypic effects on par with their cognate coding sequences. We unexpectedly discovered a family of endogenous retroviral envelope-derived proteins translated in adult myeloid cells. Among these, SYNIR is a full-length syncytin-like membrane glycoprotein that positively regulates NF{kappa}B-responsive transcription, while SEMR is a secreted protein with structural homology to the feline leukemia virus accessory protein FeLIX that drives broad transcriptional remodeling of macrophage gene programs upon knockout. Updated single-cell RNA-seq annotations and an interactive UCSC Genome Browser session integrating Ribo-seq, proteomics, and CRISPR screen data are provided as community resources. Together, these findings expand the functional landscape of noncanonical translation in immunity and establish endogenous retroviral proteins as previously unrecognized regulators of macrophage biology.

immunology↗

bioRxiv2025

CRISPRi Screen Identifies a Novel Growth Suppressor lncRNA, INSTAR, in Human Monocytes

Acute myeloid leukemia (AML) is a heterogeneous disease that arises from dysregulated myeloid proliferation. We performed a high-throughput CRISPR interference (CRISPRi) screen in the THP-1 monocytic cancer cell line to identify long noncoding RNAs (lncRNAs) that play a role in contributing to cell proliferation. Our screen identified INSTAR (Intergenic Nuclear Suppressor lncRNA Targeting Adjacent Regulator SFMBT2) as a top candidate. RNA-seq on INSTAR deficient THP-1 cells revealed transcriptional changes in genes involved in cell proliferation as well as other cellular processes. Loss of INSTAR selectively reduced expression of its neighboring gene, SFMBT2. Functional assays confirmed that both genes suppress cell growth, revealing a cis-regulatory mechanism in which INSTAR regulates SFMBT2 expression to control monocyte proliferation. Here, we leverage high-throughput screening to rapidly pinpoint functional lncRNAs providing novel insights into a key regulatory locus consisting of INSTAR and SFMBT2 which could be critical for better understanding dysregulation contributing to acute myeloid leukemia.

immunology↗

bioRxiv2025

Lifestyle-associated variation in type IV secretion systems between phytopathogenic and environmental Ralstonia

Type IV secretion systems (T4SSs) are versatile machines with variable functions including DNA uptake and release, protein translocation, and DNA conjugation. However, the diversity, distribution, and functional roles of the T4SS in the Ralstonia genus remain poorly understood. The Ralstonia solanacearum species complex (RSSC) comprises three species of plant-pathogenic bacteria that cause bacterial wilt disease. The Ralstonia genus also includes non-RSSC species that are primarily environmental bacteria and rare opportunistic human pathogens. This study compared the diversity and phylogenetic distribution of T4SSs in the RSSC phytopathogens vs. non-RSSC environmentals. Phylogenetic analysis of VirB4 sequences and synteny analysis revealed 16 distinct T4SS clusters in Ralstonia, with ten clusters found in RSSC phytopathogen genomes, twelve in non-RSSC environmental genomes, and six clusters in both groups. Collectively, these gene clusters were more prevalent in non-RSSC environmental genomes. The presence of type IV coupling protein and relaxase genes suggests that at least 14 of these T4SS gene clusters could be putative DNA conjugation systems. The clusters were encoded on accessory plasmids of various sizes or as integrative and conjugative elements (ICEs) on the chromosome or megaplasmid. The putative regions of transfer for T4SS gene clusters in the RSSC phytopathogen genomes often contained type III effectors, type VI secretion toxin/antitoxin clusters, and hemagglutinin gene clusters. In contrast, the non-RSSC environmentals were enriched in heavy metal metabolism and resistance genes. One of the 16 T4SS clusters, cluster i, exhibited evidence of specialization for the RSSC phytopathogens. These findings shed light on the eco-evolutionary differences in the Ralstonia genus. Impact statementThe Ralstonia genus contains the Ralstonia solanacearum species complex (RSSC), a group of globally important plant pathogens that impact food security. These pathogens are known to have an expansive, open pangenome with high levels of gene flow. To shed light on the eco-evolutionary differences between RSSC phytopathogens and closely related environmental species, we explored the potential role of type IV secretion systems (T4SSs) in horizontal gene flow within the Ralstonia genus. Surprisingly, these mobile genetic elements were less common in the phytopathogens than the environmentals. Nevertheless, we identified a particular T4SS gene cluster encoded on an accessory plasmid that appears to be specialized to the pathogenic lifestyle of the RSSC. This specialized cluster harbors genes that drive RSSC host range (type III effectors) and others that likely allow these pathogens to antagonize the plant microbiota (type VI secretion toxins and hemagglutinin two partner secretion systems). Moreover, this study sheds light on the cryptic lifestyles of the poorly studied environmental Ralstonia species, revealing that their conjugative T4SSs frequently carry heavy metal metabolism and resistance genes. Overall, this study provides a foundation to investigate the functional roles of T4SS clusters and their cargo genes in the fitness of phytopathogenic and environmental Ralstonia. Data summarySupplemental Table S1 lists details of the RSSC phytopathogen, non-RSSC environmental, and Burkholderiaceae family genomes used in this study. Supplemental Table S2A is the final list of 501 VirB4 sequences collectively identified in 636 RSSC phytopathogen genomes and 143 non-RSSC environmental genomes. Supplemental Table S2B includes the annotations and NCBI accessions for every gene and protein sequence in reference clusters a-p. Supplemental Table S2C is the list of putative T4SS cargo genes identified in complete Ralstonia genomes. Supplemental Table S2D is the list of putative T4SS cargo genes identified in cluster i regions from draft and complete Ralstonia genomes. Supplemental File S1 is the multiple sequence alignment (MSA) input for HMMER, made from the 753 VirB4 protein sequences from the Burkholderiaceae family genomes. Supplemental Files S2 and S3 are PDF-format versions of the species trees displaying the presence of T4SS gene clusters in RSSC phytopathogens and non-RSSC environmentals, respectively. Supplemental File S4 is the species tree displaying the presence of RSp0179 and RSp1521 in the Ralstonia genus. Supplemental File S5 is the MSA input for FastTree 2, made from the VirB4 protein sequences from Ralstonia genomes. Supplemental Files S6-S9 are various formats of the VirB4 protein tree in Figure 1. Supplemental Files S10-S25 are the GBK files for reference clusters a-p. Supplemental File S26 is the clinker output comparing the reference clusters. Supplemental Files S27-S36 are the clinker outputs comparing the putative regions of transfer of T4SSs in complete Ralstonia genomes. Supplemental File S37 is an MSA analysis comparing RSp1521 to AcvB homologs. Supplemental File S38 is the clustered average nucleotide identity (ANI) matrix for the putative regions of transfer of cluster i T4SSs in draft and complete Ralstonia genomes. Supplemental File S39 is the clinker output comparing the cluster i regions in draft and complete Ralstonia genomes. All Supplemental Tables and Files are available through Zenodo (DOI: 10.5281/zenodo.14873518). O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/675681v2_fig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1640850org.highwire.dtl.DTLVardef@5de6f5org.highwire.dtl.DTLVardef@e605feorg.highwire.dtl.DTLVardef@1937bac_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Sequence diversity of the T4SS marker VirB4 in the Ralstonia genus. VirB4 protein sequences (n=496) were identified by a custom hidden Markov model (HMM) search, and an approximately-maximum-likelihood tree was built with FastTree 2 from a MUSCLE multiple sequence alignment (MSA) of the VirB4 sequences. Bootstrap values (0 to 1) from 1,000 resamples are shown for key nodes. The unrooted tree was visualized in iTOL and labeled using Affinity Designer. The 16 distinct T4SS gene clusters (a-p) are outlined and labeled. Circles denote clusters that are present in the RSSC phytopathogens and triangles denote clusters that are present in the non-RSSC environmentals. Colored clusters were found in RSSC phytopathogen genomes, and clusters with dashed outlines were not found in the RSSC phytopathogens. Supplemental files contain the virB4 gene list (Supplemental Table S2A), the VirB4 protein sequence MSA (Supplemental File S5), the Newick file for the tree (File S6), a PDF of the unrooted tree with branch labels (File S7), and PDFs of the rectangular tree with branch labels, without and with colors (Files S8 and S9). C_FIG This studys newly sequenced genomes, Gazipur 4 and Gazipur 5, are available on NCBI with the following accessions: GCF_049860735.1 and GCF_049860725.1, respectively. The two contigs containing the cluster j T4SS were submitted to NCBI through the Third Party Annotation (TPA) section of the DDBJ/ENA/GenBank databases with the following accessions: BK072068-BK072069. These contigs were assembled from SRA reads under the accession: SRR18649448.

microbiology↗

bioRxiv2025

VILMIR is a trans-acting long noncoding RNA that enhances the host interferon response in human epithelial cells

Long noncoding RNAs (lncRNAs) have been found to play significant regulatory roles within antiviral and immune responses. We previously identified the novel lncRNA virus-inducible lncRNA modulator of interferon response (VILMIR), that was found to broadly regulate the host transcriptional response to interferon-beta (IFN-{beta}) treatment in A549 human lung epithelial cells. Here, we investigated the mechanism by which VILMIR regulates the host interferon response in trans by identifying interacting proteins and gene regulatory networks of VILMIR. Through an RNA pull-down assay, we found that VILMIR interacted with both nuclear and cytoplasmic proteins in vitro, including the transcriptional regulators FUBP1 and PUF60 in the nucleus, as well as the antiviral proteins IFIT1 and IFIT3 and the aminoacyl-tRNA synthetases QARS1 and KARS1 in the cytoplasm. In addition, we found that overexpression of VILMIR in A549 cells resulted in an overall enhancement of host interferon response genes and identified a core set of interferon-stimulated genes that were consistently regulated by VILMIR knockdown and overexpression. Finally, we proposed several possible mechanisms by which VILMIR may interact with the identified proteins to regulate the interferon response, such as by interacting with FUBP1 and PUF60 in the nucleus to regulate host transcription in trans or by interacting with the IFIT proteins and aminoacyl-tRNA synthetases in the cytoplasm to regulate translation. IMPORTANCEDespite thousands of long noncoding RNAs (lncRNAs) being differentially expressed after immune responses and viral infections, there is very limited knowledge on their individual functions in these contexts. We previously identified a novel lncRNA, VILMIR, that was found to be an interferon-stimulated gene that regulated the host transcriptional response to interferon-beta treatment in human epithelial cells. Here, we investigated the mechanism by which VILMIR regulates the interferon response in trans. Through in vitro studies, we identified several nuclear and cytoplasmic proteins that interact with VILMIR, including proteins involved in transcriptional and translational regulation. In addition, we demonstrated that the overexpression of VILMIR results in an enhancement of host interferon response genes, supporting our hypothesis that VILMIR plays an activating role in the host interferon response. Finally, we propose several potential models for the mechanism of VILMIR, providing a foundation for the investigation of VILMIR as a novel therapeutic target in antiviral immunity.

genetics↗

bioRxiv2025

EssSubgraph improves performance and generalizability of mammalian essential gene prediction with large networks

Predicting essential genes is important for understanding the minimal genetic requirements of organisms, identifying disease-associated genes, and discovering potential drug targets. Wet-lab experiments for identifying essential genes are time-consuming and labor-intensive. Although various machine learning methods have been developed for essential gene prediction, both systematic testing with large collections of gene knockout data and rigorous benchmarking for efficient methods are very limited to date. Furthermore, current graph-based approaches require learning the entire gene interaction networks, leading to high computational costs, especially for large-scale networks. To address these issues, we propose EssSubgraph, an inductive representation learning method that integrates graph-structured network data with omics features for training graph neural networks. We used comprehensive lists of human essential genes distilled from the latest collection of knockout datasets for benchmarking. When applied to essential gene prediction with multiple types of biological networks, EssSubgraph achieved superior performance compared to existing graph-based and other models. The performance is more stable than other methods with respect to network structure and gene feature perturbations. Because of its inductive nature, EssSubgraph also enables predicting gene functions using dynamical networks with unseen nodes and it is scalable with respect to network sizes. Finally, EssSubgraph has better performance in cross-species essential gene prediction compared to other methods. Our results show that EssSubgraph effectively combines networks and omics data for accurate essential gene identification while maintaining computational efficiency. The source code and datasets used in this study are freely available at https://github.com/wenmm/EssSubgraph.

bioinformatics↗

bioRxiv2025

CTCF directly binds G-quadruplex structures to regulate genome topology and gene expression

DNA G-quadruplexes (G4s) are non-B form secondary DNA structures that are highly conserved across evolution. G4 structures occupy key regulatory sites in the mammalian genomes and are implicated in several cellular processes. However, the mechanisms by which G4s contribute to distinct facets of genome function are not well understood. Here, we conduct a proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in genomic regulators of nucleosome remodeling, paraspeckle assembly, RNA splicing, and 3D genome organization. Among the most prominent hits, we identify the genomic architectural protein, CTCF, as one of the strongest G4 binders. Building on this discovery, we perform extensive biochemical validation of CTCF-G4 interaction and identify CTCF mutants, with pronounced affinity for G4s over its consensus DNA motif. By implementing well-established approaches and developing new G4 mapping tools, we define a comprehensive catalog of genomic G4s and investigate their association with CTCF binding. Using genetic reconstitution of mouse embryonic stem cells with a G4-specific CTCF mutant, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Interestingly, our studies reveal a subset of G4-linked chromatin loop anchors that form "persistent loops" which are retained even upon CTCF depletion. Collectively, our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.

molecular biology↗

bioRxiv2025

Ancient origin of an urban underground mosquito

Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. Here we investigate a widely cited example of urban adaptation, Culex pipiens form molestus, also known as the London Underground Mosquito. Population genomic analysis of [~]350 contemporary and historical samples counter the popular hypothesis that molestus originated belowground in London less than 200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Middle East over the course of >1000 years, likely in concert with the rise of agricultural civilizations. Our results highlight the role of early human society in priming taxa for contemporary urban evolution and have important implications for understanding arbovirus transmission.

evolutionary biology↗

bioRxiv2024

CRISPRi Screen Uncovers lncRNA Regulators of Human Monocyte Growth

Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 20,000 lncRNAs annotated in the human genome we do not know the function for the majority. Here we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important in viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 (RP11-542M13.2 hereafter referred to as long noncoding RNA regulator of monocyte proliferation, LNCRMP) and the second is a lncRNA called OLMALINC (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of LNCRMP and OLMALINC from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that LNCRMP regulated proapoptotic pathways while knockdown of OLMALINC impacted genes associated with the cell cycle. Data supports both LNCRMP and OLMALINC functioning in cis to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.

immunology↗

bioRxiv2024

Identification and Functional Characterization of lncRNAs involved in Human Monocyte-to-Macrophage Differentiation

Long noncoding RNAs (lncRNAs) make up the largest portion of RNA produced from the human genome, but only a small fraction have any ascribed functions. Although the role of protein-coding genes in macrophage biology has been studied extensively, our understanding of the role played by lncRNAs in this context is still in its early stages. There are over 20,000 lncRNAs in the human genome therefore, attempting to select a lncRNA to characterize functionally can be a challenge. Here we describe two approaches to identify and functionally characterize lncRNAs involved in monocyte-to-macrophage differentiation. The first involves the use of RNA-seq to infer possible functions and the second involves a high throughput functional screen. We examine the advantages and disadvantages of these methodologies and the pipelines for validation that assist in determining functional lncRNAs.

immunology↗

bioRxiv2024

CRISPRware: an efficient method for contextual gRNA library design

We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and accounts for genetic variation, which allows allele-specific guide design on a genome-wide scale. The latter ability holds promise for the development of gene therapy in the context of gene dosing and dominant negative mutations.

bioinformatics↗

bioRxiv2024

Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton

Xanthomonas spp. employ transcription activator-like effectors (TALEs) to promote pathogenicity by activating host susceptibility (S) genes. Cotton GhSWEET10 is an S gene targeted by a TALE in an early isolate of Xanthomonas citri pv. malvacearum (Xcm), but not by recent field Xcm isolates. To understand the pathogenicity shift in Xcm and its adaptation to cotton, we assembled the whole genome and the TALE repertoire of three recent Xcm Texas field isolates. A newly evolved TALE, Tal7b, activated different GhSWEET genes, GhSWEET14a and GhSWEET14b. Simultaneous activation of GhSWEET14a and GhSWEET14b resulted in pronounced water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identified a pectin lyase as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9-based gene editing supported the function of GhSWEETs as S genes in cotton bacterial blight and the promise of disrupting the TALE-binding site in these genes to control the disease. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes simultaneously to promote pathogenicity.

plant biology↗

bioRxiv2024

Human long noncoding RNA, VILMIR, is induced by major respiratory viral infections and modulates the host interferon response

Long noncoding RNAs (lncRNAs) are a newer class of noncoding transcripts identified as key regulators of biological processes. Here we aimed to identify novel lncRNA targets that play critical roles in major human respiratory viral infections by systematically mining large-scale transcriptomic datasets. Using bulk RNA-sequencing (RNA-seq) analysis, we identified a previously uncharacterized lncRNA, named virus inducible lncRNA modulator of interferon response (VILMIR), that was consistently upregulated after in vitro influenza infection across multiple human epithelial cell lines and influenza A virus subtypes. VILMIR was also upregulated after SARS-CoV-2 and RSV infections in vitro. We experimentally confirmed the response of VILMIR to influenza infection and interferon-beta (IFN-{beta}) treatment in the A549 human epithelial cell line and found the expression of VILMIR was robustly induced by IFN-{beta} treatment in a dose and time-specific manner. Single cell RNA-seq analysis of bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients uncovered that VILMIR was upregulated across various cell types including at least five immune cells. The upregulation of VILMIR in immune cells was further confirmed in the human T cell and monocyte cell lines, SUP-T1 and THP-1, after IFN-{beta} treatment. Finally, we found that knockdown of VILMIR expression reduced the magnitude of host transcriptional responses to IFN-{beta} treatment in A549 cells. Together, our results show that VILMIR is a novel interferon-stimulated gene (ISG) that regulates the host interferon response and may be a potential therapeutic target for human respiratory viral infections upon further mechanistic investigation. IMPORTANCEIdentifying host factors that regulate the immune response to human respiratory viral infection is critical to developing new therapeutics. Human long noncoding RNAs (lncRNAs) have been found to play key regulatory roles during biological processes, however the majority of lncRNA functions within the host antiviral response remain unknown. In this study, we identified that a previously uncharacterized lncRNA, VILMIR, is upregulated after major respiratory viral infections including influenza, SARS-CoV-2, and RSV. We demonstrated that VILMIR is an interferon-stimulated gene that is upregulated after interferon-beta (IFN-{beta}) in several human cell types. We also found that knockdown of VILMIR reduced the magnitude of host transcriptional responses to IFN-{beta} treatment in human epithelial cells. Our results reveal that VILMIR regulates the host interferon response and may present a new therapeutic target during human respiratory viral infections.

immunology↗

bioRxiv2023

CRISPRi screens identify the lncRNA, LOUP, as a multifunctional locus regulating macrophage differentiation epigenetically and inflammatory signaling through a short, encoded peptide

Long non-coding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous novel non-coding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP (lncRNA originating from upstream regulatory element of SPI1 [also known as PU.1]), as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames (sORFs) capable of being translated and are responsible for LOUPs ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.

immunology↗

bioRxiv2023

The RNA binding protein, HNRNPA2B1, regulates IFNG signaling in macrophages

Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) is a well known RNA binding protein but the mechanisms by which it contributes to innate immune gene regulation are poorly understood. Here we report that HNRNPA2B1 functions in macrophages to regulate IFNG (IFN-{gamma}) signaling through alternative splicing of the IFNG receptor. Specific deletion of HNRNPA2B1 in macrophages resulted in altered cytokine responses in both an endotoxic shock model and following Salmonella infection. Interestingly, while HNRNPA2B1 can function as a viability gene, we observed increased macrophage and neutrophil numbers in the KO mice following LPS induced endotoxic shock. We also discovered that HNRNPA2B1 restricts replication of Salmonella enterica in vivo. Mechanistically, loss of HNRNPA2B1 resulted in an increase in NGO transcripts, which lack a start codon, of the IFNG receptor (Ifngr) leading to lower expression of the receptor at the cell surface impacting the downstream IFNG signaling cascade. Collectively, our data highlight an important role for HNRNPA2B1 in regulating IFNG signaling and restricting intracellular bacterial pathogens in macrophages.

immunology↗

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↗

bioRxiv2022

Evolutionarily conserved effects of Notch signaling drive intestinal graft-versus-host disease in mice and non-human primates

Notch signaling promotes T-cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like ligand DLL4. To assess if Notchs effects are evolutionarily conserved and identify key mechanisms, we studied antibody-mediated DLL4 blockade in a non-human primate model similar to human allo-HCT. Short-term DLL4 blockade improved post-transplant survival with striking, durable protection from gastrointestinal GVHD, out of proportion to other disease sites. Unlike prior immunosuppressive strategies, anti-DLL4 interfered with a T-cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin a4b7 in conventional T-cells via b1 competition for a4 binding, while preserving a4b7 in regulatory T-cells. Thereby, DLL4/Notch blockade decreased effector T-cell infiltration into the gut, with increased regulatory to conventional T-cell ratios early after allo-HCT. Our results identify a conserved, biologically unique and targetable role of DLL4/Notch signaling in GVHD. One Sentence SummaryNotch signaling promotes pathogenic effector T cell infiltration of the intestine during acute graft-versus-host disease.

immunology↗

bioRxiv2021

Natural reassortment of a segmented RNA arbovirus illustrates plasticity of phenotype in the arthropod vector and mammalian host in vivo

Segmented RNA viruses are a taxonomically diverse group of 11 families that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during co-infection of a host by a process termed reassortment. Reassortment enables rapid evolutionary change, but in the case of segmented RNA viruses utilising an arthropod vector is set against the constraint of purifying selection and genetic bottlenecks imposed by replication in two evolutionarily distant hosts. In this study, we use an in vivo host: arbovirus: vector model to investigate the impact of reassortment on two phenotypic traits: vector competence and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV is comprised of 10 linear segments of dsRNA and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4 and BTV-8) were isolated from naturally infected ruminants in Europe and parental/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to infect Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the parental virus strains which allowed inferences of the genome segments conferring this phenotypic trait. IMPORTANCEReassortment between strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses with consequences for spread, persistence and impact. The ability of these pathogens to change their phenotypes rapidly in response to selection pressure in new environments presents a major challenge in understanding factors driving emergence. Utilising a natural mammalian host-insect vector infection and transmission model, we demonstrated for the first time the genetic basis for a phenotypic trait of BTV within strains directly isolated from the field and, hence, selected and relevant for natural transmission.

microbiology↗