Expression of L1 retrotransposons in granulocytes from patients with active systemic lupus erythematosus

Abstract

Background Patients with systemic lupus erythematosus (SLE) have autoantibodies against the L1-encoded open-reading frame 1 protein (ORF1p). Here, we report (i) which immune cells ORF1p emanates from, (ii) which L1 loci are transcriptionally active, (iii) whether the cells express L1-dependent interferon and interferon-stimulated genes, and (iv) the effect of inhibition of L1 ORF2p by reverse transcriptase inhibitors. Results L1 ORF1p was detected by flow cytometry primarily in SLE CD66b+CD15+ regular and low-density granulocytes, but much less in other immune cell lineages. The amount of ORF1p was higher in neutrophils from patients with SLE disease activity index (SLEDAI) > 6 (p = 0.011) compared to patients with inactive disease, SLEDAI < 4. Patient neutrophils transcribed seven to twelve human-specific L1 loci (L1Hs), but only 3 that are full-length and with an intact ORF1. Besides serving as a source of detectable ORF1p, the most abundant transcript encoded a truncated ORF2p reverse transcriptase predicted to remain cytosolic, while the two other encoded an intact full-length ORF2p. A number of genes encoding proteins that influence L1 transcription positively or negatively were altered in patients, particularly those with active disease, compared to healthy controls. Components of nucleic acid sensing and interferon induction were also altered. SLE neutrophils also expressed type I interferon-inducible genes and interferon β, which were substantially reduced after treatment of the cells with drugs known to inhibit ORF2p reverse transcriptase activity. Conclusions We identified L1Hs loci that are transcriptionally active in SLE neutrophils, and a reduction in the epigenetic silencing mechanisms that normally counteract L1 transcription. SLE neutrophils contained L1-encoded ORF1p protein, as well as activation of the type I interferon system, which was inhibited by treatment with reverse transcriptase inhibitors. Our findings will enable a deeper analysis of L1 dysregulation and its potential role in SLE pathogenesis.

Subcellular location of L1 retrotransposon-encoded ORF1p, reverse transcription products, and DNA sensors in lupus granulocytes

“…ORF1p is detectable by flow cytometry in a subset of neutrophils and low-density granulocytes from SLE patients [ 31 ]. Because the accuracy of flow cytometry for low-abundance proteins is not the best, we first quantitated ORF1p in neutrophils from SLE patients and healthy controls (HC) using an independent approach, namely a targeted mass spectrometry assay.…”

“…While the vast majority of L1 copies are truncated and/or mutated, 80–100 copies of the human-specific L1Hs subfamily are potentially retrotransposition-competent [ 28 ] and encode for two proteins, termed ORF1p and ORF2p, the latter having an N-terminal endonuclease domain and a centrally located active reverse transcriptase catalytic domain. Numerous L1Hs loci are expressed in patients with SLE [ 29 , 30 , 31 ], 7 of them full-length [ 31 ], but only 3 with an intact reverse transcriptase encoded by ORF2 [ 32 , 33 ], which during the canonical retrotransposition life-cycle of L1 reverse transcribes the L1 mRNA to create a new genomic copy if it. In addition, ORF2p can be catalytically active in the cytosol and create DNA: RNA heteroduplexes (which can give rise to ssDNA or dsDNA), even in the absence of ORF1p [ 34 , 35 ], with its own mRNA or other cellular RNAs, such as Alu transcripts, as templates [ 36 , 37 ].…”

“…Anti-ORF1p titers also correlated closely with circulating DNA in complex with neutrophil elastase or myeloperoxidase, suggesting that neutrophil death may be involved in the generation of anti-ORF1p antibodies. Indeed, ORF1p protein was detectable in neutrophils and low-density granulocytes by flow cytometry in patients with active disease [ 31 , 40 ], but much less in other immune cell lineages. The percentage of ORF1p + cells was low (0–5%) in patients with inactive disease, somewhat higher in patients with moderate disease (SLEDAI < 6; 2–20%) and considerably higher in patients with high disease activity (up to 80%).…”

Large overlap in neutrophil transcriptome between lupus and COVID-19 with limited lupus-specific gene expression

“… 12 The latter two categories of transposable elements are more active in SLE neutrophils than in healthy neutrophils. 3 13 14 …”

“…Our procedures for neutrophil isolation, RNA extraction and RNA sequencing were recently published. 3 …”