Increased expression of Siglec-9 in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a common inflammatory lung disease. Sialic acid-binding immunoglobulin-type lectins 9 (Siglec-9) is predominantly expressed on innate immune cells and has been shown to exert regulatory effect on immune cells through glycan recognition. Soluble Siglec-9 (sSiglec-9), the extracellular region of Siglec-9, might fulfill its function partly by competitive inhibiting siglec-9 binding to its ligands; however, the role of Siglec-9 and sSiglec-9 in the pathogenesis COPD remain largely unknown. In this study, we showed that Siglec-9 expression in alveolar and peripheral blood neutrophil were increased in COPD patients by immunofluorescence and flow cytometry, respectively. Plasma levels of sSiglelc-9 were elevated in COPD patients by ELISA. In vitro, Siglec-9 expression and/or sSiglelc-9 levels were up-regulated by cigarette smoke extract (CSE), lipopolysaccharide (LPS), some cytokines, and dexamethasone (DEX). Recombinant sSiglce-9 increased oxidative burst in neutrophil and enhanced neutrophil chemotaxis toward IL-8 independent on CXCR1 and CXCR2 expression, but it did not affect neutrophil apoptosis or secretions of inflammatory cytokines. In conclusion, Siglec-9 was complementarily increased to induce a negative feedback loop to limit neutrophil activation in COPD, sSiglce-9 enhanced neutrophil ROS and chemotaxis toward IL-8 likely via competitively inhibiting ligands binding to Siglec-9.

the extracellular region of Siglec-9, can prevent down-regulation of the immune responsiveness of neutrophil and provide an antibacterial benefit against Group B Streptococcus infection likely through competitively inhibiting binding of capsular polysaccharide to Siglec-9 20 . Tomioka et al. reported that sSiglec-9 exerts an antitumor function against Mucin1-expressing tumor in mice by avoiding Mucin1 binding to immune cell Siglec-9 to produce the negative immunomodulation and/or by inactivating tumor-associated Mucin1 signaling 21 .
We hypothesized that sSiglce-9 might contribute to neutrophil dysfunction in the development of COPD via competitive inhibition of Siglec-9 ligations binding to Siglec-9. Therefore, the current study was conducted to determine Siglec-9 expression on neutrophil surfaces and sSiglce-9 levels in plasma from smokers with and without COPD. In addition, we evaluated the effect of CSE, LPS, pro-inflammatory cytokines and DEX on neutrophil surface Siglec-9 expression and on sSiglce-9 levels in culture supernatant in vitro. Recombinant sSiglce-9 was applied to explore its role in regulating neutrophil function. The impact of sSiglce-9 on HBE inflammatory secretion with or without CSE and LPS was also investigated.

Results
Subject characteristics. Clinical characteristics of two group subjects providing for peripheral blood or BALF were shown in Supplementary Tables S1 and S2, respectively. In both of them, the average age and smoking history of COPD patients were similar to those of controls. Compared with COPD patients, controls exhibited significantly higher levels of FEV1 (p < 0.05), FEV1% of predicted (p < 0.01), and FEV1/FVC% (p < 0.01).
Increased Siglec-9 expression in alveolar neutrophil in COPD patients. Siglec-9 expression in lung tissue was assessed by immunohistochemistry. Neutrophil and other inflammatory cells showed positive Siglec-9 staining in human alveolar spaces (Fig. 2a).
In order to determine the expression of Siglec-9 in alveolar macrophage and neutrophil, we performed dual immunofluorescence staining for Siglec-9 and CD68 (marker of macrophage) to detect macrophage and carried out H&E staining in corresponding slides to localized neutrophil. As shown in Fig. 2b and c, both alveolar macrophage and neutrophil expressed Siglec-9. Siglec-9 expression in alveolar neutrophil was markedly increased in COPD patients relative to controls. But no difference was detected in Siglec-9 expression in alveolar macrophage between two groups (Fig. 2b). To further confirm that results, the expression of Siglec-9 mRNA in alveolar macrophage was assessed by real-time PCR. Consistent with the immunofluorescence results, Siglec-9 mRNA expression was similar in COPD patients and controls (Fig. 2d).
As CXCR1 and CXCR2 play a critical role in neutrophil migration 23 , we evaluated whether their expression was changed under sSiglec-9 incubation for 2 h. Unfortunately, sSiglec-9 had no effect on their expression (Fig. 6c,d).

Discussion
In this study, we showed that neutrophil Siglec-9 expression and sSiglelc-9 levels in plasma were increased in COPD patients. In vitro, CSE and LPS-induced neutrophil Siglec-9 expression were likely through TNF-α and IL-8; sSiglec-9 was likewise elevated by CSE and LPS presumably via TNF-α, IL-6, and IL-8, whereas DEX only augmented neutrophil Siglec-9 expression but not sSiglec-9 levels in supernatant. As an additional functional verification, we found that sSiglce-9 increased oxidative burst in neutrophil and enhanced neutrophil chemotaxis . Neutrophil Siglec-9 expression and sSiglelc-9 levels in culture supernatant were up-regulated by CSE and LPS. Neutrophils were isolated from peripheral blood of controls and then incubated for 12 h in the absence or presence of CSE or LPS at different concentrations. Following culture, neutrophil Siglec-9 expression was quantified by flow cytometry analysis, and sSiglelc-9 levels in culture supernatant were determined by ELISA. (a,c) Siglec-9 and sSiglec-9 expression were up-regulated after 0.5% CSE treatment. (b,d) LPS increased Siglec-9 and sSiglec-9 expression in a concentration-dependent manner. Data are displayed as means ± SEM; n = 15. P values were calculated using paired t tests; *p < 0.05, **p < 0.01, ***p < 0.001. toward IL-8. Taken together, Siglec-9 was complementarily increased to inhibit neutrophil activation in COPD patients and in cell model incubation with CSE and LPS. Instead, sSiglce-9 levels was higher in COPD patients than controls and subsequently enhanced neutrophil ROS and chemotaxis toward IL-8 probably via competitive inhibiting ligands binding to Siglec-9. Furthermore, DEX might exert an anti-inflammatory role through up-regulating Siglec-9 expression.
Neutrophil Siglec-9 expression was shown to be involved in septic shock and rheumatoid arthritis patients 10 . We found that Siglec-9 expression of alveolar and peripheral blood neutrophil were significantly increased in COPD patients. As Siglec-9 played a role in inhibiting immune response, so compensated elevation of Siglec-9 expression might exert a negative feedback loop to limit neutrophil responses. Besides, plasma sSiglelc-9 levels were significantly higher in COPD patients than controls.
Soluble Siglec-9 could prevent down-regulation of the immune responsiveness of neutrophil and provide an antibacterial benefit against Group B Streptococcus infection by competitively inhibiting binding of capsular polysaccharide to Siglec-9 on neutrophil surfaces 20 . Tomioka et al. reported that sSiglec-9 exerted an antitumor effect against Mucin1-expressing tumors by avoiding Mucin1 binding to immune cell Siglec-9 resulting in negative immunomodulation and/or by inactivating tumor-associated Mucin1 signaling 21 . Therefore, it was a reasonable assumption that sSiglec-9 might block the interaction of Siglec-9 with its ligands to disturb Siglec-9 function in COPD. This hypothesis was applied to understand why Siglec-9 and sSiglelc-9 are increased in the excessive and uncontrolled neutrophilic inflammatory airway disease 24,25 . More specifically, Siglec-9 was of compensatory elevation in COPD and increased levels of sSiglec-9 might prevent Siglec-9 from activation via competitive inhibition of ligands binding to Siglec-9.
CS is the main risk factor for developing COPD 26 which is accelerated through exacerbations caused by infection 27 . Consistent with previous studies showing that LPS increases Siglec-9 or Siglec-E expression 28, 29 , we found . Effect of cytokines on Siglec-9 and sSiglec-9 expression. Neutrophils were isolated from peripheral blood of controls and incubated for 12 h in the absence or presence of recombinant cytokines. Following culture, neutrophil Siglec-9 expression was quantified by flow cytometry analysis, and sSiglelc-9 levels in culture supernatant was determined by ELISA. (a,b) Siglec-9 expression was up-regulated after incubation with recombinant TNF-а and IL-8. (c,d) Levels of sSiglec-9 in culture supernatant were increased after stimulation with recombinant cytokines. Data are displayed as means ± SEM; n = 15. P values were calculated using paired t tests; *p < 0.05, **p < 0.01, ***p < 0.001. Figure 5. Dexamethasone augmented neutrophil Siglec-9 expression but not sSiglec-9 levels in culture supernatant. Neutrophils were isolated from peripheral blood of controls and incubated for 12 h in the absence or presence of dexamethasone. Following culture, neutrophil Siglec-9 expression was quantified by flow cytometry analysis, and sSiglelc-9 levels in culture supernatant was determined by ELISA. (a) 10 −4 M DEX augmented Sigled-9 expression. (b) DEX had no effect on sSiglec-9 levels in culture supernatant. Data are displayed as means ± SEM, n = 15. P values were calculated using paired t tests; *p < 0.05. that LPS induced Siglelc-9 expression in peripheral blood neutrophil, it turned out to be truth for CSE. Besides, CSE and LPS could induce sSiglec-9 expression in the culture supernatant. Thus, CSE and LPS partially accounted for higher expression of Siglec-9 and sSiglec-9 in COPD.

Figure 7.
No effect of sSiglec-9 on neutrophil apoptosis and inflammatory cytokine secretion. (a) High levels of CSE (2.5%) induced apoptosis of neutrophils isolated from peripheral blood of controls, whereas 10 ng/ml LPS reduced apoptosis. No effect of sSiglec-9 on neutrophil apoptosis were detected through the binding of annexin V-FITC and PI. Both 0.5% CSE and 10 ng/ml LPS increased neutrophils secretion of IL-6 (b), IL-8 (c) and TNFα (d) but none were affected by sSiglec-9. Data are displayed as means ± SEM, n = 15. P values were calculated using paired t tests; *p < 0.05, **p < 0.01, ***p < 0.001. CSE and LPS resulted in up-regulated production of pro-inflammatory cytokines which were involved in the pathogenesis of COPD 30 . We found that TNF-α and IL-8 increased both Siglec-9 and sSiglec-9 expression whereas IL-6 only augmented sSiglec-9 production. Therefore, TNF-α and IL-8 might attribute to CSE-and LPS-induced Siglec-9 expression. In addition, TNF-α, IL-6, and IL-8 might account for CSE-and LPS-induced sSiglec-9 production.
Leukocyte migration is critical to maintaining host defense but uncontrolled cellular infiltration into tissues can lead to chronic inflammation 31 . One key feature of COPD is excessive neutrophilic airway inflammation 24,25 . The accumulation of neutrophil can be due to increased neutrophil recruitment 32 , delayed spontaneous apoptosis 33 , or repression of the clearance of apoptotic neutrophil by tissue macrophages 34 . IL-8 is elevated in the airways of COPD patients and was one of the chemotactic factors generated at the site of inflammation. It is a powerful mediator of neutrophil migration and activation 35,36 . Our results found sSiglce-9 enhanced neutrophil migration toward IL-8 but had no influences on apoptosis. It was reasonable to postulate that high levels of sSiglce-9 partly contributed to excessive neutrophilic airway inflammation in COPD by elevating neutrophil recruitment. Siglec-9 and Siglec-E are important negative regulators of neutrophil and dendritic cells recruitment 12,13 . Therefore, sSiglce-9 increased neutrophil chemotactic response probably through binding to Siglec-9 ligands to block Siglec-9 activation.
IL-8 activates neutrophil via CXCR1 and CXCR2; the latter appears to be the predominant receptor mediating its chemotactic response 23 . Circulating neutrophil CXCR1 expression is significantly higher in COPD patients than in controls 37 . We detected that neutrophil CXCR1 and CXCR2 expression were not changed after incubation with sSiglec-9 for 2 h; therefore, sSiglec-9 enhanced neutrophil chemotaxis toward IL-8 was independent of CXCR1 and CXCR2 expression. The precise mechanisms of neutrophil chemotactic response toward IL-8 requires further investigation.
Considerable evidence links COPD with increased oxidative stress 38 . Circulating neutrophil from cigarette smokers and patients with exacerbations of COPD exhibit more oxidant burden 39 , possibly due to the fact that cigarette smoke contains more than 1014 oxidants per puff 40 and that infection and air pollutants have the potential to produce oxidative stress 41 . Although engagement of Siglec-9 or Siglec-E suppressed oxidative stress 14,42 , we observed that sSiglce-9 significantly boosted levels of oxidative burst in neutrophil in the present study. This observation may be due to sSiglce-9 competitive inhibiting ligands of siglec-9 binding to siglec-9.
Neutrophil apoptosis is associated with inflammatory diseases and appears to be regulated by neutrophil survival cytokines, such as granulocyte/macrophage colony-stimulating factor (GM-CSF) 43 . Several in vitro experiments have shown that Siglec-9 engagement with antibodies causes neutrophil death, especially in some cytokine-primed cells 10,44 . GM-CSF has been considered to be a survival factor for neutrophil but GM-CSF was showed to promote death of neutrophil in association with Siglec-9 cross-linking when neutrophil was preincubated with GM-CSF and subsequently Siglec-9 stimulated. When GM-CSF was added at the same time or after the addition of an anti-Siglec-9 antibody, the cytokines had no effect 10 . The mechanisms underlying the interplay of neutrophil survival cytokines and Siglec-9 are complex and not completely resolved. In our study, spontaneous neutrophil apoptosis was not altered by sSiglec-9 stimulation; high level CSE-induced and LPS-reduced neutrophil apoptosis were also not affected, possibly due to the interplay of neutrophil survival cytokines and Siglec-9.
Matsubara et al. observed only a combination of monocyte chemoattractant protein-1 and sSiglec-9, but not alone, repaired spinal cord injury by anti-inflammatory M2-like macrophage induction 45 . However, Matsumoto et al. reported that sSiglec-9 alone alleviated the joint inflammation and destruction in a collagen-induced arthritis by suppression of M1 proinflammatory macrophage, which led to lower levels of TNF-a and IL-6 46 . Contrary to their results, we found that sSiglec-9 had no effect on neutrophil or smooth muscle cell (data not shown) secretion of inflammatory cytokines but enhanced LPS-induced HBE secretion of IL-8 which might subsequently recruit more neutrophil into the lungs. Thus, this result highlighted that sSiglec-9 might induce a positive feedback loop to aggregate airway inflammation in COPD by enhancing IL-8 production under infection. The impacts of sSiglec-9 on cytokine secretion appear to be cell type-specific or dependent on cytokines in the circumstance.
Corticosteroids, anti-inflammatory drugs, are therapeutic agents for relieving COPD symptoms 22 . Siglec-5/14 expression is significantly higher in sputum cells from COPD patients receiving inhaled corticosteroids than those without them 16 . DEX is a synthetic corticosteroid and has been widely used for the treatment of COPD. In the current study, we found that DEX augmented neutrophil Siglec-9 expression but not sSiglec-9 levels in culture supernatant. These results indicate that DEX might exert an anti-inflammatory effect on neutrophil by up-regulating Siglec-9 expression.
In conclusion, Siglec-9 was complementarily increased to serve as a negative feedback loop to limit neutrophil activation in COPD. Soluble Siglce-9 was higher in COPD patients and subsequently enhanced neutrophil ROS and chemotaxis toward IL-8 likely via competitively inhibition of ligands binding to Siglec-9. Furthermore, DEX might exert an anti-inflammatory role through up-regulating Siglec-9 expression.

Subjects.
A total of 51 COPD patients and 23 healthy controls were included in our study. The clinical characteristics of them are described in Supplementary Table S1. The COPD patients were recruited from outpatient of the Tongji hospital between April 2014 and April 2016. COPD was diagnosed according to the criteria established by the NHLBI/WHO Global Initiative for COPD 47 . Control subjects were selected from a pool of healthy men who visited the general health check-up center in the Tongji hospital. Subjects were excluded if they suffered from asthma, other obstructive lung diseases, tumors or a course of oral corticosteroids in the previous 4 weeks. 15 Supplementary Table S2. Subjects with history of asthma, allergy, pneumonia, interstitial lung diseases and bronchiectasis were excluded.
Lung tissues specimens were collected during surgical resection of solitary pulmonary nodules as described previously 48 .
This study was conducted in compliance with the institutional policy regarding the protection of patients' private information and approved by the Ethical Committee of Tongji Hospital, Huazhong University of Science and Technology (IRB ID: 20140415). Written informed consent was obtained from all participants. Sample collection and all the experimental methods in our study were carried out in accordance with the approved guidelines.
Isolation and culture of neutrophil. Neutrophil was isolated from peripheral blood by Ficoll-Hypaque gradient centrifugation as described previously 49 . Briefly, peripheral blood was mixed with equal volume of hydroxyethyl starch 550 (TBDscience, Tianjin, China) and PBS, and allowed to sediment for 30 min at room temperature. The leukocyte containing supernatant was then carefully layered onto gradient reagent (TBDscience, Tianjin, China). After centrifugation at 800 g for 25 min, all the layers without polymorphonuclear (PMN) were removed. This method routinely yielded a purity >98% as determined by Wright-Giemsa staining. The viability of isolated cells was found to be greater than 96% with trypan blue (Sigma, St. Louis, USA) exclusion. After washing in phosphate-buffered saline solution, both cells were suspended at concentration of 5 × 106 cells/mL in 1640 RPMI medium (GIBCO Laboratories, Grand Island, NY) in 6-well plates (Nest, China).

BALF collection and isolation of Bronchoalveolar Macrophage. During fiberoptic bronchoscopy,
BALF was collected prior to any other procedures according to international guidelines 50 . The bronchoscope was wedged in the subsegmental bronchus of the middle lobe not containing any nodule. The bronchus was lavaged with 50 ml aliquots of sterile saline solution at 37 °C and then the fluid was aspirated. Two further 50 ml aliquots of saline solution were instilled and aspirated in the same way. Aliquots were transferred to 50 ml polypropylene tubes on ice.
BALF was strained through a 40 μm cell strainer (Millipore, Germany) and then centrifuged at 500 × g for 10 min at 4 °C. The cell pellet was resuspended in 2 ml RPMI 1640 and total cell viability was determined by trypan blue exclusion. Slides were prepared by cytocentrifugation at 500 × g for 5 min. slide were stained with a standard May-Grunwald Giemsa stain and a differential count was performed by examining 300 cells. Other slides were fixed by 4% formaldehyde for immunofluorescence.
Bronchoalveolar macrophage were allowed to adhere to sterile 6-well polystyrene culture plates (Costar, NY, USA) at 37 °C for 2 h. Nonadherent cells were removed by washing the monolayers with PBS, yielding monolayers that contained at least 95% macrophages by morphologic criteria. 1 ml TRIzol was added for extracting total RNA.

Cell stimulation.
To determine the siglec-9 expression on neutrophil surface, cells were stimulated with different concentration of CSE 51 , LPS (sigma-Aldrich, St. Louis, USA), TNF-α, IL-6 and IL-8 (Peprotech, Rocky Hill, UAS) for indicated time. In order to determine the effect of sSiglec-9 on neutrophil or bronchial epithelial cells (HBE) (ATCC, VA, USA) cytokines secretion, cells were pretreated with recombinant siglec-9 (sSiglec-9) (R&D Systems, Minneapolis, USA) for 1 h, and then treated with CSE or LPS for indicated time. Supernatants were stored at −80 °C for ELISA analysis, and cells were collected for detection. Flow cytometry. After treatment, cells were washed and resuspended in 300 μl Cell Staining Buffer (Biolegend, San Diego, USA). Cells were incubated with the appropriate primary antibodies for 30 minutes in the dark. Primary antibodies are as following: Human Siglec-9 Fluorescein (FITC)-conjugated antibody and IgG2A isotype control-FITC were obtained from R&D Systems (Minneapolis, USA); Human CXCR1 FITC-conjugated antibody, IgG2B isotype control-FITC, Human CXCR2 phycoerythrin (PE)-conjugated antibody as well as IgG1 isotype control-PE were obtained from Biolegend (San Diego, USA). Samples were washed twice in ice-cold PBS and resuspended in 300 μl Cell Staining Buffer (Biolegend, San Diego, USA), and then assessed by flow cytometry using a Becton Dickinson LSR flow cytometer 48 .

Intracellular ROS detection. Intracellular ROS level was detected using Reactive Oxygen Species Assay Kit
(Beyotime, Jiangsu, China). Neutrophil was pretreated with sSiglec-9 or recombinant IL-8 for 2 h. After treatment, cells were washed and incubated in RPMI 1640 medium with 10 uM dichlorofluorescein diacetate (DCFH-DA) for 20 min. Subsequently, the cells were washed 3 times with the RPMI 1640 medium without serum. Finally, the cells were resuspended with 300 μl PBS, and the mean fluorescent signal intensity (MFI) of dichlorofluorescein (DCF) was determining by flow cytometry.

Assessment of apoptosis.
An annexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGEN, Nanjing, China) was used for detection of neutrophil apoptosis 49 . Immunofluorescence and H&E staining. After rehydration in a graded alcohol series, slides of BALF were washed in TBS and heated at 100 °C for 10 min in sodium citrate buffer for epitope retrieval. Slides were incubated with blocking reagent (Dako Japan Ltd., Kyoto, Japan) for 1 h at room temperature, and then were incubated overnight with anti-CD68 antibody (Abcam, Cambridge, UK) plus anti-Siglec-9 antibody (R&D Systems, Minneapolis, USA) or matched isotype controls (Becton Dickinson) at 4 °C. After washing, samples were incubated with Alexa Fluor 594-goat anti-mouse and Alexa Fluor 488-donkey anti-goat secondary antibody (Invitrogen, California, USA) for 60 min at room temperature. Nuclei were counterstained with DAPI. Subsequently, H&E staining were carried out in the slides which were double immunostaining for Siglec-9 and CD68.
Neutrophil chemotaxis assays. The activity of neutrophil migration was estimated through a polycarbonate filter with 3-μm pore size in 24-well transwell chambers (Costar, NY, USA). Neutrophil was incubated with sSiglec-9 for 2 hours in RPMI 1640 medium supplemented with 2% heat-inactivated FCS. Lower compartments were filled with 500 μl RPMI 1640 medium with or without 50 ug/ml IL-8 (Peprotech, Rocky Hill, USA). Then, 200 μl neutrophil suspension (1 × 10 6 /ml) was added to upper chamber and incubated for 1 hour. Cells on the lower surface of the filters and in the lower chamber were harvested in 300 μl PBS and counted by flow cytometry acquiring events for a fixed time period of 60 seconds 53, 54 . Enzyme linked immunosorbent assay (ELISA). Concentrations of IL-6, IL-8 and TNF-а in cell-free culture supernatants or sSiglec-9 in plasma were measured using DuoSet ELISA kits (R&D Systems, Minneapolis, USA) as described previously 49 . The limits of detection for the IL-6, IL-8, TNF-а and sSiglec-9 ELISA kits were 9.38, 31.3, 15.6 and 62.5 pg/mL, respectively. Statistical analyses. Data were presented as means ± SEM. Statistical analyses were performed using Prism