Abstract Coxiella burnetii (Cb), the causative agent of Q fever, replicates within host macrophages by modulating immune responses through poorly understood mechanisms. Long non-coding RNAs (lncRNAs) are crucial yet underexplored regulators of inflammation, particularly in Cb pathogenesis. Employing a comparative transcriptomic analysis of THP-1 macrophages infected with 16 different microbes, we dissect a core set of immune-responsive lncRNAs such as MAILR, LINC01215, PACER, and MROCKI-common to human anti-pathogen responses, and distinguish them from lncRNAs specifically altered at early (1 h) time points in individual infections. In particular, our approach identifies lncRNA CYP1B1-AS1 as specifically upregulated in a spatiotemporal manner along with CYP1B1 in cis during Cb infection. Promoter assays confirm their co-regulation via a shared bidirectional promoter, while aryl hydrocarbon receptor (AHR)-lucia luciferase and nuclear translocation assays demonstrate that Cb infection activates AHR, driving their transcription. Knockdown of CYP1B1-AS1 or CYP1B1 alone disrupts mitochondrial homeostasis, increases ROS and mitochondrial dysfunction, and exacerbates apoptosis during infection. These findings position the CYP1B1-AS1/CYP1B1 axis as a key regulator of mitochondrial homeostasis under AHR signaling, supporting an intracellular environment that benefits Cb replication. Our results highlight the critical roles of lncRNAs in immune regulation and provide a valuable resource for future lncRNA research. Subject terms: Pathogens, Mechanisms of disease __________________________________________________________________ In this work, authors utilise comparative transcriptomics to reveal lncRNAs that distinguish pathogen-specific from core macrophage responses. They identify a Q fever-specific AHR-regulated CYP1B1-AS1/CYP1B1 axis that modulates mitochondrial homeostasis and survival of Coxiella burnetii. Introduction Coxiella burnetii (Cb), the causative agent of Q fever, is a highly adapted obligate intracellular Gram-negative pathogen that primarily infects monocytes and macrophages. It establishes a replicative niche within lysosome-matured Coxiella-containing vacuoles (CCVs)^[45]1, a niche that is non-permissive to most intracellular pathogens^[46]2. Cb utilizes a type IVB secretion system (T4SS) to translocate effectors into host cells, thereby manipulating endocytic trafficking, immune evasion, to promote intracellular replication^[47]3–[48]5. The Nine Mile strain of Cb, commonly used in research, exists in two variants: phase I (NMI, virulent), expressing a full-length lipopolysaccharide (LPS), and phase II (NMII, avirulent), characterized by truncated LPS^[49]6. Infection typically occurs via inhalation, causing acute flu-like illness, but chronic cases can lead to severe complications like endocarditis and lung fibrosis^[50]7. Long-term antibiotics are the standard treatment, highlighting the need for new therapies and diagnostics. A hallmark of Cb infection is its ability to evade innate immune activation distinguishing it as a stealth pathogen^[51]1,[52]8,[53]9. In contrast, other well-characterized intracellular pathogens like Mycobacterium tuberculosis^[54]10, Legionella pneumophila^[55]11, and Salmonella enterica subsp. enterica serovar Typhimurium^[56]12, actively engage cytosolic sensors such as cGAS, NLRs, and TLRs, triggering a robust inflammatory response^[57]13,[58]14. Despite significant research efforts, the molecular mechanisms by which Cb evade these immune activations remain poorly understood. Long non-coding RNAs (lncRNAs) are regulatory transcripts over 200 nucleotides, and have emerged as important regulators of inflammation and immune responses^[59]15,[60]16. Macrophages are the first line of immune defense, recognizing pathogen-associated molecular patterns (PAMPs), such as LPS, via pattern recognition receptors (PRRs) to activate central innate immune signaling pathways involving NF-κB and type I interferon pathways (IFN-I) against intracellular infections^[61]17. Recent comprehensive lncRNA transcriptome studies in human macrophages have revealed the significant involvement of lncRNAs such as MAILR, LINC01215, LUCAT1, PIRAT1, [62]AC010980, LINC00158 and MROCKI in regulating NF-κB and IFN-I response during infection^[63]17–[64]19. Other well-characterized lncRNAs, such as NEAT1, GAPLINC, HOTAIR, PACER, and LINC-COX2, are induced upon LPS stimulation, and infections caused by M. tuberculosis, S. Typhimurium, HCV and HIV-1, promoting pro-inflammatory response and pathogen restriction^[65]20,[66]21. Despite these discoveries, most lncRNAs expressed in human immune cells remain functionally uncharacterized, likely due to their complex molecular interactions and context-dependent expression patterns, which hinder mechanistic investigation. The involvement of lncRNAs in Cb infection has not been explored. Considering Cb’s unique immune evasion strategy and its tropism for macrophages, we hypothesized that Cb elicits a distinct lncRNA expression profile compared to other bacterial infections. To test this, we employed a comparative multi-pathogen RNA-sequencing (RNA-seq) strategy in THP-1-derived macrophages infected with 16 different microbes. This systems-level strategy identified a core set of lncRNAs commonly responsive to any pathogen confrontation (herein termed as immune-responsive lncRNAs) and distinguished them from lncRNAs uniquely altered in individual infections. Notably, this strategy identified lncRNAs specifically regulated during Cb infection, and provided insights into their temporal dynamics in host defense. Among these, CYP1B1-AS1 emerged as a key lncRNA upregulated during Cb infection. Our in vitro studies demonstrated that CYP1B1-AS1 is co-regulated with its neighboring gene CYP1B1 via a shared bidirectional promoter. Transcriptional assays and luciferase reporter analyses revealed that this co-regulation is mediated by activation of the transcription factor (TF)-aryl hydrocarbon receptor (AHR). Functional analyses showed that silencing of CYP1B1-AS1 or CYP1B1 resulted in mitochondrial dysfunction, elevated reactive oxygen species (ROS), and apoptosis exacerbated by Cb infection. Furthermore, silencing either of the genes augmented pro-inflammatory cytokine production, implicating this axis in maintaining mitochondrial homeostasis and regulating ROS-associated inflammation. By establishing the CYP1B1-AS1/CYP1B1 axis as an AHR-regulated, infection-responsive module, our study provides new insights into host-Coxiella interactions. Importantly, the comparative transcriptomic framework provides a scalable platform for identifying lncRNA in human macrophages and serves as a valuable resource for future investigations into immunity and infection. Results Identification of infection- and immune-responsive lncRNAs in human macrophages To investigate lncRNA expression profiles across different bacterial infections and distinguish pathogen-specific responses from common macrophage immune programs, we conducted a comprehensive comparative transcriptomic analysis. Phorbol 12-myristate-12 acetate (PMA)-differentiated THP-1 cells (THP-1 macrophages) were infected for 1 h with a panel of 16 bacterial strains, including three C. burnetii Nine Mile Phase II (Cb; BL2 variant as WT), and two attenuated mutants dotA::Tn (CbA/ΔdotA), and dotB::Tn (CbB/ΔdotB), alongside 13 additional bacterial species with varying pathogenicity and non-pathogenic controls (Supplementary Data [67]1). These included (a) Pathogenic bacteria: Enterohemorrhagic Escherichia coli O157 (EcT; EHEC), Enterohemorrhagic Escherichia coli O157 Δstx (nontoxigenic) (EcN; EHECΔstx), Francisella novicida U112 (Fn), Pseudomonas aeruginosa PAO1 (Pa), Staphylococcus aureus JE2 (Sa), S.Typhimurium (STm), Brucella melitensis ΔvjbR (Bmv), and (b) Opportunistic pathogen: Enterococcus faecalis (Ef), Rhizobium radiobacter (Rr), Micrococcus luteus (Ml), and (c) three non-pathogenic controls: Escherichia coli DH5α (Ec5), Listeria innocua (Li), and Bacillus subtilis P31K6 (Bs). The sample preparation, RNA sequencing, and data analysis workflow is outlined in Supplementary Fig. [68]1a, b. Considering Cb’s stealth-like infection strategy and poor innate immune activation, we hypothesized that it would evoke a distinct lncRNA expression profile compared to other pathogens. To evaluate this, we implemented a three-step analysis pipeline: (a) RNA-seq data were used to identify commonly differentially expressed (DE) lncRNAs across ≥4 infections, designated as immune-responsive lncRNAs; (b) we extracted DE-lncRNAs specific to individual infections, which serve as pathogen-specific lncRNAs;(c) comparative analyses were conducted to identify DE-lncRNAs uniquely induced or absent during Cb infection at 1 h post-infection (p.i.). We identified 2348 DE-lncRNAs (log[2] fold-change (FC) ≥ 1 or ≤−1; p < 0.05) across all infection conditions compared to uninfected (Mock) controls (Supplementary Data [69]2). Principal component analysis (PCA) revealed 33.98% variance between mock and infected macrophages, with 14.42% variance across infection groups (Fig. [70]1a). An UpSet analysis (Supplementary Fig. [71]1c) was performed to identify immune-responsive lncRNAs and pathogen-responsive lncRNA (Fig. [72]1b). Differential transcriptomic profiles across all infections are illustrated in Supplementary Fig.[73]2a–m. Fig. 1. RNA-seq analysis reveals common and pathogen-specific immune-regulatory lncRNA expression profiles in THP-1 macrophages across bacterial infections. [74]Fig. 1 [75]Open in a new tab a Principal component analysis (PCA) of RNA-seq data illustrating transcriptional variance across infections, including C. burnetii Nine Mile Phase II (Cb), C. burnetii Nine Mile Phase II dotA::Tn (CbA), C. burnetii Nine Mile Phase II dotB::Tn (CbB), Escherichia coli DH5α (Ec5), enterohemorrhagic E. coli O157 (EcT/EHEC), E. coli O157Δstx (EcN/EHECΔstx), Bacillus subtilis P31K6 (Bs), Francisella novicida U112 (Fn), Pseudomonas aeruginosa PAO1 (Pa), Staphylococcus aureus JE2 (Sa), Salmonella enterica subsp. Typhimurium SL1344 (STm), Rhizobium radiobacter (Rr), Micrococcus luteus (Ml), Listeria innocua (Li), Enterococcus faecalis (Ef), and Brucella melitensis ΔvjbR (Bmv). PCA was performed using normalized RNA-seq data to assess global transcriptional variance. Infected samples are denoted by circles; mock-infected controls as triangles. Data points are color-coded by infection. b Experimental workflow for identifying common and pathogen-specific lncRNAs following infection of THP-1 macrophages. Schematics created using BioRender.com. c Bar graph summarizing the number of pathogen-specific differentially expressed (DE) lncRNAs identified across the infections analyzed. d Heatmap showing quantitative real-time PCR (RT-qPCR) validation of selected immune-regulatory lncRNAs that are either commonly regulated across infections or specifically altered during C. burnetii infection. Expression normalized to ACTB and shown relative to mock-infected controls (set to 1). lncRNAs with a log₂ fold change ≥ 1.5 or ≤ 0.5 were considered DE. Data represent mean ± SD from three independent experiments (n = 3). Source data are provided as a Source Data file. We identified 538 immune-responsive lncRNAs (Supplementary Data [76]3). Additionally, we identified 19, 208, and 165 DE-lncRNAs specific to Cb, CbA, and CbB infections, respectively (Fig. [77]1c). No significant DE-lncRNAs were detected in Ef- or Bmv-infected transcriptomes, likely due to low infectivity or delayed host response at this early time point, and therefore, we excluded them from further analysis. Among the 538 common immune-responsive lncRNAs, several well-characterized immune-regulatory lncRNAs were identified, including MAILR, LINC01215, LUCAT1, MROCKI, MIR155HG, MIR222HG, PACER, and EGOT (Table [78]1, Supplementary Data [79]3). These lncRNAs have recently been shown to be induced in primary human macrophages upon microbial infection or pattern recognition receptor (PRR) stimulation^[80]17,[81]19,[82]22–[83]24. For instance, MAILR promotes TRIF-IRF3 signaling by stabilizing Optineurin, enhancing IFN-β production. LUCAT1 suppresses immune activation via its interaction with hnRNPs and STAT1. LINC01215 and MAILR are robustly induced by L. pneumophila infection and LPS exposure, while MROCKI enhances inflammatory responses by repressing GATA2 and promoting NF-κB signaling in macrophages^[84]17,[85]23. To validate their expression, we performed quantitative real-time PCR (RT-qPCR) analysis, which confirmed that the selected immune-responsive lncRNAs were induced across bacterial infections, in agreement with the RNA-seq findings. Notably, MAILR, LINC01215, MROCKI, and PACER were significantly upregulated upon L. pneumophila infection, which served as a positive control. This observation corroborates prior reports describing their early induction (1 h post LPS-challenge) and confirms their activation in L. pneumophila-infected macrophages (Fig. [86]1d). Boxplot analysis summarizing their expression trends across infections is shown in Supplementary Figs. [87]2n–o and [88]3a–d. Table 1. Representative immune-responsive lncRNAs regulated across multiple infections Immune-responsive lncRNAs Other disease/pathogenic infection No. of pathogens DE in this study References