Background/Purpose: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoantibody production and periods of elevated disease activity. Recent studies indicate that along with dysfunctional adaptive immune responses, neutrophils are also important in disease pathogenesis. SLE patients have higher numbers of low-density granulocytes (LDGs) in peripheral blood that express neutrophil markers and are hyper-responsive to external stimuli. LDGs are suggested to be pathogenic in lupus. However, the mechanisms leading to increased LDGs in SLE patients are unclear.

Methods: Peripheral blood mononuclear cells from 19 SLE patients with varying disease activity and 11 controls were enriched by density gradient centrifugation. LDG numbers were determined by flow cytometry. Plasma cytokines were measured by xMAP assays. Induced pluripotent stem cells (iPSC) derived from adult peripheral blood CD34+ cells were differentiated into neutrophils using feeder-culture conditions. Cultures were supplemented with IL-13 at different stages of differentiation: day 7 (hemangioblasts-like cells to CD34+ hematopoietic stem cells (HSC)), day 12 (HSC into CD45+ hematopoietic cells), day 18 (CD45+ cells into neutrophil precursors), day 23 (maturation to segmented neutrophils).

Results: SLE patients with elevated disease activity had significantly higher frequency of immature LDGs as determined by surface marker expression (p=0.005). IL-13 levels were higher in SLE patients with elevated disease activity compared to low disease activity patients (p=0.01) and controls (p=0.006), and the levels correlated negatively with absolute neutrophil numbers (r = -0.4404, p= 0.0354). Our goal was to test whether abnormal timing and excessive IL-13 exposure of hematopoietic stem cells (HSC) leads to generation of immature neutrophils in SLE patients due to defective differentiation. The iPSC cultures were induced into neutrophilic differentiation. By day 23 of differentiation, presence of IL-13 reduced frequencies of neutrophilic cells. The presence of IL-13 did not inhibit early stages of neutrophil differentiation. However, the frequencies of more mature band and segmented cells was significantly reduced when exposed to IL-13 at day 7. In the cultures where IL-13 supplementation was started at day 18 and day 23, neutrophils were generated when allowed to differentiate for a longer duration (Fig 1). Thus, although delayed, maturation was restored with late IL-13 exposure. Although IL-13 inhibited neutrophilic differentiation, it did not affect the generation of CD34+ HSC or CD45+ cells.

Conclusion: Our data suggest that SLE patients may have dysregulated development of myeloid cells in the bone marrow due to the cytokine milieu during elevated disease activity. Abnormal timing and exposure to IL-13 may increase immature neutrophils in SLE patients, possibly by altering the differentiation potential of CD34+ HSC. The functional responses of these newly generated neutrophils may differ due to differences in the bone marrow transit time. The dysregulation of granulopoiesis therefore may generate neutrophils that further exacerbate autoimmune response.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-sle-associated-cytokines-in-generation-of-mature-neutrophils/