
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12249-9
10.1016/j.heliyon.2024.e36218
e36218
Research Article
Increased circulating LOX-1+ neutrophils activate T cells and demonstrate a pro-inflammatory role in allergic rhinitis
Deng Xiao-Hui dengxh8@mail2.sysu.edu.cn
dengxiaohui94@163.com
ab
Huang Long-Xin 13480396592@163.com
ab
Sun Qi- 693264779@qq.com
ab
Li Chan-Gu lcgyjy123@163.com
ab
Xie Ying-Chun chunyx1214@163.com
ab
Liu Xiao-Qing liu_xq0963@163.com
ab
Fu Qing-Ling fuqingl@mail.sysu.edu.cn
ab⁎
a Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, PR China
b Department of Allergy, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, PR China
⁎ Corresponding author. Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, PR China. fuqingl@mail.sysu.edu.cn
16 8 2024
15 9 2024
16 8 2024
10 17 e3621817 1 2024
9 8 2024
12 8 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
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.

Graphical abstract

Schema for increased circulating LOX-1+ neutrophils activate T cells and demonstrate a pro-inflammatory role in allergic rhinitis. Graphical abstract

Image 1

Highlights

• LOX-1+ neutrophils were significantly elevated in the PB of patients with AR.

• LOX-1+ neutrophils derived from patients with AR promoted T cell proliferation and pro-inflammatory cytokine production.

• IgE, age, and sex did not affect LOX-1+ neutrophil levels.

Keywords

Allergic rhinitis
LOX-1+ neutrophils
Pro-inflammatory role
Low-density neutrophils
==== Body
pmc1 Introduction

Allergic rhinitis (AR) is a global concern and a growing public health, medical, economic, and social problem worldwide with a prevalence of up to 50 % in some regions [1,2]. AR is caused by immunoglobulin E (IgE)-mediated reactions to inhaled allergens and is characterized by nasal itching, sneezing, nasal rhinorrhea, and congestion. Although AR is not typically life threatening, its detrimental effects cannot be ignored. AR is often trivialized, and the quality of life of patients with AR can be significantly affected, including, but not limited to, impaired work productivity and classroom performance, disturbed sleep, and heavy psychological burden [3,4]. Therefore, effective therapies for AR are an important unmet clinical need.

Low-density neutrophils are a heterogeneous population with anti-inflammatory (such as polymorphonuclear myeloid-derived suppressor cells [PMN-MDSC]) or pro-inflammatory (such as low-density granulocytes [LDG]) properties [5,6]. Notably, LDG and PMN-MDSC share overlapping neutrophil surface markers used for identification, including CD11b+, CD15+, CD33+, CD14−, and human leukocyte antigen-DR isotype (HLA-DR)- [[6], [7], [8], [9]]. Lectin-type oxidized LDL receptor-1 (LOX-1), a 50 KDa transmembrane glycoprotein, is co-expressed in pro-inflammatory and suppressive low-density neutrophils and has been identified as a specific marker of PMN-MDSC in cancer [[10], [11], [12]]. Immune cells involved in AR processing and regulation, including T helper cells (Th), T regulatory cells (Tregs), group 2 innate lymphoid cells (ILC2), and dendritic cells (DCs), have attracted extensive attention and have been well described [[13], [14], [15], [16], [17], [18], [19], [20]]. However, the role of CD14−CD11B+CD15+LOX-1+ neutrophils (LOX-1+ neutrophils) in AR remains unclear.

In the present study, we investigated LOX-1+ neutrophils and their effects in patients with AR. To the best of our knowledge, our study is the first to determine that these neutrophils are significantly elevated in patients with AR, and have a pro-inflammatory role.

2 Materials and methods

2.1 Participants

In this study, 25 patients with AR (female, n = 10; male, n = 15; average age, 25.6 years) and nine healthy individuals (female, n = 7; male, n = 2; average age, 24 years) were enrolled between May 2021 and September 2023. The diagnosis of AR was in accordance with the criteria of Allergic Rhinitis and Its Impact on Asthma (ARIA) guidelines [21]. The patients presented with nasal itching, sneezing, nasal congestion, and rhinorrhea. The subjects were excluded if pregnant, patients with allergic dermatitis. The subjects have not taken glucocorticoids or received the other treatments (eg, antihistamines or immunotherapy) for 4 weeks before the beginning of the study. This study was approved by the Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University (No.2019-331), and informed consent was obtained from all participants.

2.2 Flow cytometry analysis

Peripheral blood (PB) samples were analyzed within 6 h of sampling. Blood samples were treated with red blood lysis buffer (64010-00-100; BioGems). Anti-human antibodies of CD14-PE-CYN7 (61D3, 25-0149-42, Invitrogen), CD11B-FITC (ICRF44, 11-0118-42, Invitrogen), CD15-EF450 (HI98, 48-0159-42, Invitrogen), LOX-1-APC (15C4, 358606, Biolegend) and its corresponding isotype control-APC (MOPC-173, 400222, Biolegend), and Fixable Viability Dye eFluor™ 506 (eBioscience, 65-0866-14) were used. For the LOX-1+ neutrophil phenotype, CD14−CD11B+CD15+LOX-1+ was obtained from live whole blood cells. Cell phenotypes were analyzed using flow cytometry using the CytoFLEX S system (BECKMAN), and data were analyzed using FlowJo V10.8.1 software.

For flow cytometry sorting, a BD FACSAria III system (BD Biosciences) was used. Peripheral blood samples were analyzed within 6 h of sampling and treated with red blood lysis buffer. The isolated cells were co-incubated with anti-human antibodies of CD14-PE-CYN7, CD15-EF450 and LOX-1-APC/its corresponding isotype control-APC at 4 °C for 30 min. Subsequently, LOX-1+ neutrophils were sorted using CD14−CD15+LOX-1+ sorting from these stained cells.

2.3 CD3+ T cell isolation

Peripheral blood mononuclear cells (PBMC) were isolated from fresh PB using Lymphocyte Separation Medium (LSM; 50494X-10, Cytiva) according to the manufacturer's instructions. The isolated PBMC were washed with phosphate-buffered saline (PBS) to remove residual medium, and CD3+ T cells were isolated from PBMC using human CD3 MicroBeads (Miltenyi, 130-050-101), according to the manufacturer's instructions.

2.4 T cell proliferation and activation assay

T cell proliferation and activation assay were performed following previous studies [10,22,23]. Briefly, T cell proliferation was determined using the CellTrace™ carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Invitrogen, C34570). Purified CD3+ T cells were stained with CellTrace™ CFSE (1:1000 dilution), according to the manufacturer's instructions, and then stimulated with anti-CD3 (5 μg/mL) coated on plates and soluble anti-CD28 (1 μg/mL). Cells were then cultured alone or with LOX-1+ neutrophils at different rations (T: LOX+ NEUT 1:0, 2:1, 1:1 or 1:2) for 3 d. After 3 d of culture, cells were stained for anti-human antibodies CD4-APC-Cy7 (RPA-T4, 561839, BD Bioscience) and CD8-FITC (SK1, 344703, Biolegend) and then analyzed using the CytoFLEX S system. The supernatants were collected and stored at −80 °C. Anti-CD3 (UCHT1, 14-0038-82) and anti-CD28 (CD28.2, 16-0289-81) antibodies were purchased from eBiosciences.

2.5 Enzyme-linked immunosorbent assay (ELISA) to determine levels of cytokines

Levels of pro-inflammatory and Th2 cytokines, IL-1β, IL-4, IL-5, IL-6, IL-13 and TNF-α, in cell culture supernatants and plasma were determined using the IL-1β Human Uncoated ELISA Kit (88-7261-22, ThermoFisher), IL-4 Human Uncoated ELISA Kit (88-7046-88, ThermoFisher), IL-5 Human Uncoated ELISA Kit (88-7056-88, ThermoFisher), IL-6 Human Uncoated ELISA Kit (88-7066-22, ThermoFisher), IL-13 Human Uncoated ELISA Kit (88-7439-88, ThermoFisher), and TNF-α Human Uncoated ELISA Kit (88-7346-22, ThermoFisher), respectively. All procedures were performed according to the manufacturer's instructions.

2.6 Schematic diagram

Schematic diagram was performed using https://app.biorender.com/.

2.7 Statistical analyses

Statistical analyses were performed using GraphPad software. Statistical significance was determined using an unpaired t-test, ordinary one-way ANOVA and linear regression. Statistical significance was set at P < 0.05.

3 Results

3.1 Circulating LOX-1+ neutrophils were significantly increased in patients with AR

To investigate the potential role of LOX-1+ neutrophils in AR, we first evaluated the number of LOX-1+ neutrophils in the PB of patients with AR using flow cytometric analysis. Circulating LOX-1+ neutrophils were defined by CD14−CD11B+CD15+LOX-1+ in live whole blood cells as showed in Fig. 1A. We determined that the frequencies of LOX-1+ cells in polymorphonuclear neutrophils (PMN) were significantly increased in patients with AR when compared with those from healthy donors (HD) (Fig. 1B). Moreover, the results showed that both the frequencies and absolute cell counts of LOX-1+ neutrophils in the PB were significantly increased in patients with AR compared to HD (Fig. 1C and D). These results indicate that LOX-1+ neutrophils in the PB were significantly increased in patients with AR.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. 1

3.2 LOX-1 expression of neutrophils from patients with AR was significantly elevated

LOX-1 is a specific marker of PMN-MDSC in cancer, and is expressed in both pro-inflammatory and suppressive low-density neutrophils. Flow cytometry was performed to determine the expression of LOX-1 in neutrophils. We determined that, compared to HD, the mean fluorescence intensity (MFI) of LOX-1 in neutrophils derived from patients with AR was significantly elevated (Fig. 2A and B).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. 2

3.3 LOX-1+ neutrophils from patients with AR promoted T cell proliferation

Low-density neutrophils exert either pro-inflammatory or suppressive effects. To elucidate the effects of LOX-1+ neutrophils in patients with AR, a T cell proliferation assay was performed. CFSE-labeled CD3+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies cultured alone or at the indicated ratios of LOX-1+ neutrophils. Stimulation with anti-CD3 and anti-CD28 antibodies resulted in elevated proliferation of CD3+, CD3+CD4+, and CD3+CD8+ T cells compared to the negative control group. Interestingly, when compared to CD3+ T cells cultured alone, the addition of LOX-1+ neutrophils from patients with AR significantly promoted the proliferation of T cell types (Fig. 3A–D). These results indicate that LOX-1+ neutrophils may play a pro-inflammatory role in AR.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. 3

3.4 LOX-1+ neutrophils induced a pro-inflammatory T cell cytokine profile

To further explore the effects of LOX-1+ neutrophils on T cell cytokine production, cell culture supernatants were analyzed for different cytokines using ELISA. IL-13 is one of the most important cytokines in AR [24]. Firstly, we observed higher levels of IL-13 in the plasma in AR patients compared to those in healthy controls (Fig. 4A). We determined that the addition of LOX-1+ neutrophils from patients with AR induced significantly higher production of pro-inflammatory cytokines, TNF-α and IL-6 (Fig. 4B). IL-1β production was elevated, as shown using statistical analysis, but this elevation was not statistically significant (Fig. 4B). For Th2 cytokines, IL-4 and IL-13 levels were significantly elevated in the T cells and LOX-1+ neutrophils co-culture supernatants (Fig. 4C). IL-5 production was elevated as well, but this elevation was not statistically significant (Fig. 4C). Overall, the phenotypic and functional analyses of these cells indicated that LOX-1+ neutrophils represent a pro-inflammatory subset of AR.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. 4

3.5 LOX-1+ neutrophil levels were not affected by IgE levels, age, and sex

Clinicopathological analysis was performed to gain a thorough understanding of LOX-1+ neutrophils. IgE is routinely examined in patients with AR, who we divided into two groups based on total IgE (tIgE) levels (<120 UI/mL and ≥120 UI/mL) and compared the LOX-1+ neutrophil levels between these two groups. There were no significant differences between the two groups (Fig. 5A). Moreover, we compared LOX-1+ neutrophil levels between allergen-specific IgE (sIgE)-negative (0) and-positive (1) patients with AR and elucidated that there was no significant difference (Fig. 5B). These results indicate that IgE may not affect LOX-1+ neutrophil levels in patients with AR. Furthermore, the number of LOX-1+ neutrophils was not related to age (Fig. 5C) and sex (Fig. 5D). Overall, these data revealed that IgE level, age, and sex did not affect the percentage of circulating LOX-1+ neutrophils.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.

Fig. 5

4 Discussion

AR is an increasingly prevalent global health, medical, and social problem. Recently, significant progress has been made to further understand the involvement of immune cells such as Th2, ILC2, Tregs, B cells, B regulatory cells (Breg), DCs, T follicular helper cells (Tfh), and follicular regulatory T cells (Tfr) in AR progression and regulation [[25], [26], [27], [28]]. However, the role of LOX-1+ neutrophils in AR remains unclear. In this study, the levels and role of circulating LOX-1+ neutrophils in patients with AR, as well as factors that influence them, were investigated.

LDG and PMN-MDSC are low-density neutrophils that share overlapping surface markers. AR is often associated with asthma. Previous preclinical and clinical studies have reported elevated circulating LDG levels in asthma both in preclinical and clinical research [29,30]; therefore, we hypothesized that there may be variations in low-density neutrophils in patients with AR. Circulating CD14−CD11B+CD15+LOX-1+ cells were defined as PMN-MDSC in cancers. In this study, we determined that LOX-1+ neutrophils were significantly upregulated in the whole blood of patients with AR using percentages and absolute cell counts, which was in accordance with data from patients with asthma and horses. The expression of LOX-1, one of the primary receptors for oxidized low-density lipoproteins (oxLDL), on immature neutrophils is positively associated with coronavirus 2019 (COVID-19) severity and thromboembolic complications in critical patients with COVID-19 [31]. The expression of LOX-1 in polymorphonuclear neutrophils from patients with AR was significantly increased.

LOX-1+ neutrophils suppress T cell immune responses in cancer and chronic inflammation, such as nasopharyngeal carcinoma, glioblastoma multiforme, hepatocellular carcinoma, and necrotizing enterocolitis [23,[32], [33], [34]]. Interestingly, these cells were reported for the first time to induce a pro-inflammatory T cell response in systemic lupus erythematosus [22]. We determined that LOX-1+ neutrophils from patients with AR significantly promoted T cell proliferation, including CD3+, CD3+CD4+, and CD3+CD8+ cell levels, in contrast to data on multiple cancers reported by previous studies. Interestingly, our results showed that cytokines, both pro-inflammatory cytokines (TNF-α and IL-6) and Th2 cytokines (IL-4 and IL-13), were elevated in T cells and LOX-1+ neutrophils co-culture supernatants. These results revealed that LOX-1+ neutrophils play a pro-inflammatory role in AR and reaffirmed that LOX-1 should not be used alone to assess whether neutrophils are pro-inflammatory or immunosuppressive.

AR is an IgE-mediated, type 2 inflammatory disease. Both tIgE and sIgE tests are routine tests for AR. Interestingly, LOX-1+ neutrophil levels were not affected by either type of IgE in this study. This may be attributed to the insufficient detection (insufficient allergen detection). Furthermore, we elucidated that both age and sex did not influence LOX-1+ neutrophil levels. Overall, LOX-1+ neutrophil levels were not affected by IgE, age, or sex in our study; however, and the actual influencing factors remain to be explored.

We acknowledge that there are some limitations of this study. Although we showed that LOX-1+ neutrophils can induce T cell proliferation and pro-inflammatory cytokine production, these were performed by in vitro experiments. Detailed effects of LOX-1+ neutrophils in the pathogenesis of AR should be further determined in vivo using animal models and/or nasal mucosa from the patients. In addition, we did not include more experiments about the interaction between LOX-1+ neutrophils and T cells by cell-cell contact or a paracrine effect, and more data to support that LOX-1+ neutrophil levels were not affected by IgE, age, or sex. Finally, large sample size should be considered to fully elucidate the roles of LOX-1+ neutrophils in AR and investigate of the roles of these cells in other inflammatory model system like atopic dermatitis in the future.

5 Conclusion

In conclusion, we determined that LOX-1+ neutrophils were significantly upregulated in the whole blood of patients with AR. Unlike PMN-MDSC, these cells are involved in inducing T cell proliferation and production of pro-inflammatory cytokines. We excluded IgE, age, and sex as factors influencing LOX-1+ neutrophil levels. Our findings provide new insights into the role of LOX-1+ neutrophils in AR.

Ethical statement

This study was approved by The Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University (No.2019-331).

Consent for publication

All authors agreed to publication of the work.

Disclosure of potential conflicting interests.

All authors declared no conflicts of interests.

Funding

This work was supported by grants from the 10.13039/501100001809 National Natural Science Foundation of China (No. 82271144 and 81970863 ), 10.13039/501100021171 Guangdong Basic and Applied Basic Research Foundation (2021B1515120062 ), and Guangzhou Key R&D Program (2023B03J1233 ).

Availability of data and materials

Data supporting the findings of this study are available upon reasonable request from the corresponding authors. The data are not publicly available because of privacy and ethical restrictions.

CRediT authorship contribution statement

Xiao-Hui Deng: Writing – original draft, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Long-Xin Huang: Project administration, Investigation. Qi- Sun: Data curation. Chan-Gu Li: Project administration, Investigation. Ying-Chun Xie: Project administration, Investigation. Xiao-Qing Liu: Project administration, Investigation. Qing-Ling Fu: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

AR allergic rhinitis

CFSE carboxyfluorescein succinimidyl ester

ELISA enzyme-linked immunosorbent assay

HD healthy donors

IgE immunoglobulin E

LDG low-density granulocytes

LOX-1 lectin-type oxidized LDL receptor-1

LOX-1+ neutrophils CD14-CD11B+CD15+LOX-1+ neutrophils

MFI mean fluorescence intensity

NEUT neutrophils

ns no significant

PB peripheral blood

PBMC peripheral blood mononuclear cells

PMN polymorphonuclear neutrophils

PMN-MDSC polymorphonuclear myeloid-derived suppressor cells

sIgE allergen-specific IgE

tIgE total IgE

Acknowledgments

We are extremely grateful to the entire team of the Division of Allergy, First Affiliated Hospital, Sun Yat-sen University, including medical workers, graduate students, and volunteers.
==== Refs
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