Background/Purpose:

Neutrophils are innate immune cells that play a central role in the initiation of inflammatory arthritis as well as mediating tissue damage. We sought to improve our understanding of the role of neutrophils in joint inflammation using two multiplex discovery modalities. We characterized simultaneous circulating and transmigrated, joint-derived neutrophils from patients with inflammatory arthritis and blood from healthy controls via mass cytometry (CyTOF) and RNA-Seq with the goal of gaining new insights into the biology of neutrophils in inflamed environments.

Methods:

We established a biospecimen pipeline that allowed for both immediate processing as well as cryopreservation of fresh samples. Synovial fluid and simultaneous peripheral blood samples were obtained from seven consented patients with inflammatory joint disease. Purified neutrophils were cryopreserved and stained with metal-conjugated antibodies directed against 30 surface proteins including lineage markers, adhesion molecules, chemokine receptors, Fc- and complement receptors and immune checkpoints. Gene expression of freshly isolated neutrophils from inflammatory arthritis patients and healthy controls was examined via Smart-seq2. Each gene was modeled as a linear-mixed combination of donor- and group-specific effects using DESeq2.

Results:

Circulating and transmigrated neutrophils demonstrated fundamentally distinct gene- and surface protein expression. Joint-derived neutrophils exhibited an activated phenotype reflected by increased surface expression of integrins, adhesion molecules and chemokine receptors and increased transcription of complement receptor, TNF and CD69 among many others. Intriguingly, joint neutrophils overexpressed the adaptive immunity regulatory gene PD-L1 – confirmed by CyTOF – suggesting that they might also act to restrain pathological inflammation by negatively regulating T-cell function. We further identified 11 significantly enriched Gene Sets in joint neutrophils compared to blood, including TNF-α signaling, IFN-α response, IL-2 and IL-6 signaling. Confirmation studies are ongoing.

Conclusion:

Using multiplex discovery modalities, this study examined both circulating and joint-derived neutrophils in the context of joint inflammation. The dysregulated pathways identified suggest that neutrophils not only serve as effectors of immunity but also provide feedback that could potentially regulate adaptive immune responses.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/multiplexed-characterization-of-circulating-and-joint-derived-human-neutrophils-in-inflammatory-arthritis/