Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• naRNA

• LL37

• NETs

Main Findings​

Neutrophils eliminate pathogens via secretion of antimicrobial proteins, phagocytosis, and release of extracellular traps (NETs).Notably, a NET is composed of decondensed chromatin and antimicrobial proteins such as LL37. Although NETs have important roles in host defense, they appear to exacerbate inflammation in several non-infectious diseases such as psoriasis and rheumatoid arthritis (1).Nonetheless, the exact mechanisms granting proinflammatory properties to NETs are not fully understood. Interestingly, RNA was recently shown to be a common component of NETs (2), and two independent groups have recently shown that self-RNA in complex with LL37 can activate dendritic cells (DCs) (3) and neutrophils (2), respectively. In this preprint, Bork et al. use RNA-specific antibodies and click-labeled nucleic bases to demonstrate that NET-associated RNA (naRNA) is a common component of NETs regardless of stimuli. In addition, they use RNA-sequencing to show that naRNA contains more non-ribosomal RNA than the corresponding total RNA isolated from neutrophils. Importantly, they use the TLR8 inhibitor CU-CPT9a and TLR13-deficient mice to confirm that naRNA drives NET self-propagation via TLR8 in humans and TLR13 in mice, respectively. Furthermore, they build on their previous findings to show that naRNA can activate other cell types besides neutrophils such as a macrophage cell line, as well as primary and immortalized keratinocytes. The authors also inject ears of wildtype and TLR13-deficient mice with 1) NETs(generated in the presence of RNAse inhibitors) to confirm that naRNA can induce inflammation in a TLR13-dependent manner, and 2) imiquimod (a TLR7 agonist) to show that RNA-sensing via TLR13 is necessary to drive psoriasis-like skin inflammation.

Finally, the authors use confocal microscopy and Pearson’s colocalization analysis to demonstrate that LL37 is complexed with self-RNA within neutrophils prior to stimulation.

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Limitations

• As most of the findings presented in this preprint replicate previous results published by the same group, it is difficult to identify the novelty of this new manuscript.

• The potential roles of naRNA+LL37 in host defense and sterile inflammation are two topics that undoubtedly deserve to be investigated. Given that this manuscript focuses on the role of naRNA+LL37 as a DAMP, perhaps it would be wiser to leave any findings regarding its potential role in host defense for a separate manuscript.

• The authors propose that, given the limited lifespan of extracellular RNA,RNA+LL37 complexes might have time-dependent effects. Testing this hypothesis would be of great interest to the field and could also enhance the novelty of the present manuscript.

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Significance/Novelty

Here, the authors confirm that naRNA is a common component of NETs and that it drives NET propagation via TLR8 and TLR13 in humans and mice, respectively. Moreover, they demonstrate that naRNA can activate immune and structural cells in vitro and drive inflammation in vivo. These findings offer a mechanistic explanation to the pro-inflammatory properties of NETs.

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References​

(1) Fousert, E., Toes, R. & Desai, J. Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses. Cells. 9(4):915 (2020).

(2) Herster, F., Bittner, Z., Archer, N. K. et al. Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis. Nat. Commun.11(1):105 (2020).

(3) Ganguly, D., Chamilos, G., Lande, R. Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8.J. Exp. Med. 206(9):1983-1994 (2009)

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Credit

Reviewed by Eduardo I. Cardenas as part of the cross-institutional journal club of the Immunology Institute of the Icahn School of Medicine, Mount Sinai (U.S.A.), the Kennedy Institute of Rheumatology, University of Oxford (U.K.), and the Center for Molecular Medicine, Karolinska Institutet (Sweden). You can follow him on Twitter.