CYP1B1-AS1 regulates CYP1B1 to promote Coxiella burnetii pathogenesis by inhibiting ROS and host cell death

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 dissected a core set of immune-responsive lncRNAs such as MAILR, LINC01215, PACER, and MROCKI-common to human anti-pathogen responses, and distinguished them from lncRNAs specifically altered at early (1 h) time points in individual infections. In particular, our approach unveiled lncRNA CYP1B1-AS1 as specifically upregulated in a spatiotemporal manner along with CYP1B1 in cis during Cb infection. Promoter assays confirmed their co-regulation via a shared bidirectional promoter, while aryl hydrocarbon receptor (AHR)-lucia luciferase and nuclear translocation assays demonstrated that Cb infection activates AHR, driving their transcription. Knockdown of CYP1B1-AS1 or CYP1B1 alone disrupted mitochondrial homeostasis, increased ROS and mitochondrial dysfunction, and exacerbated 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. THP-1 monocytes were differentiated into macrophages using Phorbol 12-myristate 13-acetate (PMA, 100 ng/mL, 48 h) before infection with various bacterial strains. Infections were carried for 1 hour, after which cells were collected for RNA extraction. Bacterial Strains used,Pathogenic strains: Coxiella burnetii Nine Mile Phase II (NMII), Coxiella burnetii NMII dotA::Tn (T4SS mutant), Coxiella burnetii NMII dotB::Tn (T4SS mutant), Enterohemorrhagic Escherichia coli (O157, O157 Δstx), Francisella novicida U112, Pseudomonas aeruginosa PAO1, Staphylococcus aureus JE2, Salmonella enterica subsp. Typhimurium SL1344, Rhizobium radiobacter, Micrococcus luteus, Enterococcus faecalis, Brucella melitensis ΔvjbR. Non-pathogenic controls: Escherichia coli DH5α, Listeria innocua, Bacillus subtilis P31K6. Total RNA was extracted using TRIzol™ reagent (Invitrogen) following manufacturer protocols. RNA-seq libraries were generated using the TruSeq Stranded Total RNA-Seq Library Kit (Illumina), incorporating rRNA depletion. cDNA synthesis was performed using random primers, and paired-end libraries were constructed following the manufacturer’s instructions. Differentially expressed long non-coding RNAs (lncRNAs) and associated mRNAs were identified using DESeq2, comparing C.burnetii-infected samples to control and other microbial infections.