Neutrophils are innate immune cells that drive the progression of rheumatoid arthritis (RA) through the release of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and proteases that damage host tissues. Neutrophil activation is regulated, in part, by dynamic changes in gene expression. In this study we have used RNAseq to measure the transcriptomes of neutrophils from people with severe, methotrexate-refractory RA and healthy controls. We identified a dynamic gene expression profile in people with severe RA. This is dominated by a type-I interferon-induced gene expression signature as well as activation of genes regulating neutrophil degranulation, NET production, response to ROS and oxidative stress. Whilst we did not detect significantly elevated levels of interferon-alpha in RA blood sera, we identified increased expression in RA neutrophils of miR-96-5p and miR-183-5p which regulate activation of the interferon pathway as members of the miR-183C cluster. We also detected significantly elevated NET debris in RA blood sera (p<0.05). Using gene set variation analysis we explored the heterogeneity of neutrophil gene expression in RA and identified subsets of patients with gene expression profiles reflecting enhanced neutrophil degranulation and cytotoxicity, tissue inflammation or activation by interferons. Comparison with published single-cell RNAseq datasets identified RA transcriptomes where neutrophils were polarised by genes relating to early or late cell maturity, with significant genes in each polarised state being regulated by miR-146a-5p, miR-155-5p, miR-183-5p or miR-96-5p. Overall our study demonstrates the heterogeneity of the RA neutrophil transcriptome and proposes miRNA-driven mechanisms for regulating the activated neutrophil phenotype in RA.

The authors have declared no competing interest.

This study was funded by two fellowships from Versus Arthritis (No. 19437 and 21430), a research award from Illix Limited (UK), and a Wellcome Trust ISSF award (No. 204822/z/16/z).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was approved by the University of Liverpool Central University Research Ethics Committee C for healthy controls (Ref: 1672), and NRES Committee North West (Greater Manchester West, UK) for people with RA (Ref: 11/NW/0206). All participants gave written, informed consent in accordance with the declaration of Helsinki.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors