Increased circulating LOX-1+ neutrophils activate T cells and demonstrate a pro-inflammatory role in allergic rhinitis

Abstract

Background: Low-density neutrophils are heterogeneous immune cells with immunosuppressive (such as polymorphonuclear myeloid-derived suppressor cells [PMN-MDSC]) or pro-inflammatory (such as low-density granulocytes [LDG]) properties that have been well described in multiple cancers and immune diseases. However, its role in allergic rhinitis (AR) is still unclear.

Methods: In the present study, we defined low-density neutrophils as CD14^-CD11B⁺CD15⁺LOX-1⁺ (LOX-1⁺ neutrophils), and their levels in the peripheral blood (PB) were evaluated and compared between patients with AR and healthy donors using flow cytometric analysis. LOX-1 expression on polymorphonuclear neutrophils was identified. Carboxyfluorescein succinimidyl ester (CFSE)-stained CD3⁺ T cells were cultured alone or with LOX-1⁺ neutrophils, T cell proliferation was assessed using flow cytometry, and pro-inflammatory cytokines in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA). Clinicopathological analyses were performed to gain a thorough understanding of LOX-1⁺ neutrophils.

Results: We determined that LOX-1⁺ neutrophils were significantly increased in the PB of patients with AR, and LOX-1 expression in neutrophils from patients with AR was elevated. Interestingly, LOX-1⁺ neutrophils derived from patients with AR, unlike PMN-MDSC, promoted T cell proliferation and pro-inflammatory cytokine production. Moreover, clinicopathological analysis revealed that there was no any relation between circulating LOX-1⁺ neutrophil levels and the levels of IgE, age and sex.

Conclusion: These findings indicate that elevated circulating LOX-1⁺ neutrophils play a pro-inflammatory role in AR.

Keywords: Allergic rhinitis; LOX-1+ neutrophils; Low-density neutrophils; Pro-inflammatory role.

Graphical abstract

Fig. 1

Circulating LOX-1⁺ neutrophils were increased in patients with AR. (A) Gating strategy of LOX-1⁺ neutrophils using flow cytometry analysis in healthy donors (HD) and patients with AR (AR). (B) Percentages of LOX-1⁺ cells in PMN. (C) Percentages of LOX-1⁺ neutrophils in PB. (D) Absolute cell counts of LOX-1⁺ neutrophils in 1 mL PB. Nine healthy donors and 19 patients with AR were enrolled. In all plots, mean with standard deviation (SD) are shown. *p < 0.05, **p < 0.01 using the unpaired t-test.

Fig. 2

LOX-1 expression was significantly elevated in neutrophils from patients with AR. (A) Representative flow cytometric analysis of MFI of LOX-1 expression in CD15⁺ neutrophils. (B) Statistical analysis of LOX-1 MFI in HD and AR groups. Nine healthy donors and 19 patients with AR were enrolled. In all plots, means with SD are shown. **p < 0.01 using the unpaired t-test.

Fig. 3

LOX-1⁺ neutrophils from patients with AR promoted T cell proliferation. CD3⁺ T cells isolated from PBMC were stained with CFSE, stimulated with anti-CD3 and anti-CD28, and cultured alone or with LOX-1⁺ neutrophils for 3 d. (A) Representative flow cytometric analysis of T cell proliferation. (B) Statistical analysis of CD3⁺ T cell proliferation. (C) Statistical analysis of CD3⁺CD4⁺ T cell proliferation. (D) Statistical analysis of CD3⁺CD8⁺ T cell proliferation. NEUT, neutrophils. n = 3–6. Means with SD are shown for all plots. **p < 0.01, ***p < 0.001, ****p < 0.0001 using the ordinary one-way ANOVA.

Fig. 4

LOX-1⁺ neutrophils activated pro-inflammatory cytokine expression. Cytokine levels in the plasma and the cell culture supernatants were determined by ELISA. (A) Statistical analysis of IL-13 levels in the plasma (n = 9). (B–C) A T/LOX-1⁺ NEUT 1:1 ratio was used in this experiment. (B) Statistical analysis of pro-inflammatory cytokine (TNF-α, IL-6 and IL-1β) levels in the supernatants (n = 5). (C) Statistical analysis of Th2 cytokine (IL-4, IL-5 and IL-13) levels in the supernatants (n = 5). Means with SD are shown for all plots. *p < 0.05, **p < 0.01 using the unpaired t-test.

Fig. 5

LOX-1⁺ neutrophil levels could not be affected by IgE levels, age and sex. (A) Statistical analysis of LOX-1⁺ neutrophils in low (<120 IU/mL) (n = 8) and high (≥120 IU/mL) (n = 10) tIgE (total IgE) group in patients with AR. (B) Statistical analysis of LOX-1⁺ neutrophils in 0 (negative) (n = 10) and 1 (positive) (n = 9) sIgE (specific IgE) group in patients with AR. (C) Linear regression of LOX-1⁺ neutrophils and age (n = 28). (D) Statistical analysis of LOX-1⁺ neutrophils in female (n = 15) and male (n = 13) participants. In all plots, means with SD are shown; ns, no significant, using the unpaired t-test.