Background/Purpose:

Polymorphonuclear neutrophils (PMN) are abundant and activated in rheumatoid arthritis (RA) joints. In addition to their pro-inflammatory role, PMN exert immunoregulatory functions and may thus affect T-cell responses. Regulatory T lymphocytes (Treg) play a key role in the control of autoimmunity but they are functionally deficient in RA. Very few data are available on Treg-PMN communication in normal conditions, and even less during inflammation, especially in RA. The aim of this study was to analyze the mechanisms controlling the interaction between Tregs and PMNs and the consequences on their activity.

Methods:

Splenic Treg and bone marrow PMN (C57BL/6 mice) were purified by magnetic sorting. Treg and PMN of healthy donors and RA patients were freshly isolated by magnetic sorting from peripheral blood. Co-cultures were unstimulated or exposed to anti-CD3/anti-CD28 antibodies and/or LPS. CD4⁺FoxP3⁺ Treg (mouse and human), Ly6G⁺ (mouse) and CD66b⁺ (human) PMN were identified by flow cytometry. Cell activation was studied using antibodies against CD39, CD25, CTLA-4 (Treg) and CD11b (PMN). Treg maintenance was evaluated as the frequency of FoxP3 expression among CD4⁺ cells, and cell proliferation by CFSE staining followed by flow cytometry analysis. In some case, co-cultures were performed using Transwell (0.4 mm). Cytokine levels were quantified in culture supernatants by ELISA. Collagen-induced arthritis (CIA) was induced by immunization with type II collagen in complete Freund’s adjuvant.

Results: Without stimulation of both Treg and PMN from naive mice, no effect on any cell type was observed in co-culture. In contrast, co-culture of activated Treg with activated PMN resulted in Treg proliferation and increased maintenance of FoxP3 expression, with higher CTLA4 but lower CD39 expression, sustained PMN activation evidenced by CD11b up-regulation, and higher secretion of MIP-2, IL-6 and IL-17 but not IFN-γ in naive mice. All these effects were lost in transwell experiments or when blocking JAK signalling. Nevertheless, transfer of supernatants from LPS-activated PMN also partly increases Treg maintenance. Most importantly, PMN sustain Treg suppressive effect on effector T cells (CD4⁺FoxP3^–) proliferation. Similar results were observed in co-cultures of activated Treg/PMN isolated from CIA mice, although in vitro activation of PMN is not required. Likewise, human Treg-PMN co-cultures led to enhanced Treg maintenance with higher expression of CTLA4/CD25 in a cell contact-dependent manner in both healthy donors and RA patients. Secretion of IL-8 and IL-10 was enhanced in co-cultures.

Conclusion:

Our results reveal the existence of a link between Treg and PMN, mainly leading to an activation of both cell types in normal or inflammatory conditions. Although cell contacts are clearly required, soluble mediators are also involved and probably as a second signal. Whether these synergistic interactions lead to a global suppressive or inflammatory milieu with functional modulation of either partner needs to be clarified, especially in RA patients.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/polymorphonuclear-neutrophils-and-regulatory-t-lymphocytes-treg-cooperate-to-sustain-treg-activity-in-normal-and-arthritic-contexts/