Decreased expression of a phagocytic receptor Siglec-1 on alveolar macrophages in chronic obstructive pulmonary disease

Alveolar macrophages serve as professional phagocytes that have the ability to eliminate inhaled microorganisms or noxious particles and endogenous apoptotic cells or cellular debris for innate host defence and the resolution of inflammation [1]. However, in the lung of patients with chronic obstructive pulmonary disease (COPD), alveolar macrophages participate in pathological processes of chronic inflammation and tissue remodelling (i.e. alveolar destruction and small airway fibrosis) by producing reactive oxygen species (ROS), pro-inflammatory cytokines/chemokines, elastolytic proteases and pro-fibrotic mediators [2, 3]. In addition to the dysregulated production of the mediators, recent studies have indicated that alveolar macrophages of COPD were functionally defective in the phagocytosis of microbial pathogens, such as Haemophilus influenzae [4‐6], Streptococcus pneumoniae [5] and Moraxella catarrhalis [6]. This defective phagocytosis of microbes is implicated in the bacterial colonisation and persistent inflammation in the airway of COPD, which are postulated to cause acute exacerbations [7].

Phagocytosis is defined as the cellular uptake of particles (> 0.5 μm) by a plasma-membrane envelope and consists of multiple biological processes: recognition, engulfment, plasma-membrane fusion, maturation of phagosomes with progressive acidification and culminating in phagolysosomal fusion and digestion [8]. Plasma membrane-receptors have a critical role in the sensing and engulfment of microbes in the initial phase of phagocytosis and are classified into (i) opsonic receptors (Fc receptors and complement receptors) and (ii) non-opsonic, pattern-recognition receptors (scavenger receptors and lectin-like recognition molecules) [9]. Scavenger receptors such as macrophage scavenger receptor-1 (MSR-1, also known as SR-A1), macrophage receptor with collagenous structure (MARCO, also known as SR-A2) and CD36 vary in the structure of their extracellular domains and recognise a large variety of molecules including not only microbial ligands (lipopolysaccharide and lipoteichoic acid) but also endogenous self-ligands (lipoproteins, unmodified proteins, etc.) [10]. Lectin-like recognition molecules also consist of diverse membrane-bound receptors such as C-type lectin receptors (mannose receptor or dectin-1, etc.) and sialic acid-binding immunoglobulin-like lectins (e.g. Siglec-1) [11]. This extremely diverse array of plasma membrane-receptors allows professional phagocytes to sense and eat a wide variety of foreign pathogens.

It is known that the expression of cell surface molecules is altered in alveolar macrophages of smokers or patients with COPD. Smoking reduces the expression of cell surface molecules involved in apoptotic cell-clearance (CD91) and cellular adhesion (CD31, CD44) [12]. Moreover molecules involved in antigen presentation (CD86 and CD11a) are down-regulated in the alveolar macrophages of COPD [13]. Despite the increasing evidence of the phenotypic alteration of alveolar macrophages under a pathological condition, the expression levels of receptors mediating bacterial phagocytosis have not been clearly determined.

Our data suggested two novel aspects for human alveolar macrophages which may explain the compromised phagocytic ability of the alveolar macrophages in COPD; (i) the extracellular expression of plasma membrane-bound receptor for phagocytosis, Siglec-1, could be altered under pathological conditions in COPD lung and (ii) Siglec-1 was at least partially involved in the engulfment of NTHi by human alveolar macrophages. Importantly we observed reduced Siglec-1 expression in mild-to-moderate COPD subjects who had never experienced exacerbations, indicating that phagocytic receptor expression could be altered in early stages of COPD.

Siglecs (sialic acid-binding immunoglobulin (Ig)-like lectins) are type 1 membrane proteins containing a homologous N-terminal V-set Ig-like domain that recognises sialylated glycan, followed by variable numbers of C2 set domains [18]. The Siglec family is classified into two subfamilies: (i) Siglecs that are conserved across mammals (Siglec-1, CD22, myelin-associated glycoprotein and Siglec-15) and (ii) CD33-related Siglecs that are variable across mammals and therefore are postulated to have evolved rapidly [17]. Most Siglecs except for Siglec-1 have cytoplasmic regions that mediate intracellular signalling and regulate innate and adaptive immunity. CD22 and most CD33-related Siglecs have immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains [18], that suppress activation signals by recruiting cytoplasmic phosphatases containing a Src homology 2 (SH2) domain [19]. Conversely a few Siglecs, such as Siglec-14, coupled with immunoreceptor tyrosine-based activating motifs (ITAMs)-bearing adaptors have the ability to activate immune cells via spleen tyrosine kinase (Syk) [17].

Some emerging evidence from human genetic studies has suggested that Siglecs are involved in pathogenesis of COPD. Null allele of SIGLEC14 encoding immune-activating Siglec-14 was associated with a reduced risk of COPD exacerbation, possibly via suppression of pro-inflammatory responses by inactivation of Syk [20]. An integrative genomics approach utilising single nucleotide polymorphisms (SNPs) on COPD phenotypes and a genome-wide expression quantitative trait loci (eQTLs) study on lung tissues revealed that CD22 was one of potential causal genes for airflow limitation in patients with COPD [21]. However, to our knowledge, there has been no report showing a relationship between Siglec-1 and COPD.

Siglec-1 (also known as sialoadhesin or CD169) preferentially binds to α2,3-linked sialic acids [22, 23] and has longer extracellular regions (i.e., 17 Ig domains) that lack intracellular signalling motifs [24]. This unique structure of Siglec-1 may enable extension of sialic acid-binding sites outside the plasma membranes [25] and probably is useful for recognition, capture and uptake of microbes [17]. Siglec-1 is expressed by tissue macrophages and has a critical role in recognition and clearance of sialylated bacterial pathogens such as Neisseria meningitidis [26], Campyrobacter jejuni [27] and group B streptococcus [28]. In addition, Siglec-1 was required for proper pro-inflammatory responses via tumour necrosis factor-α and interferon (IFN)-β to eliminate C.jejuni that was intravenously administered to mice [29]. NTHi is also known to be a sialylated microbe and has genes encoding α2,3-sialyltransferase which adds sialic acids onto its lipooligosaccharide [30, 31]. Considering a number of evidence that NTHi is one of the most common bacteria involved in bacterial colonisation and acute exacerbation of COPD [32, 33], our data suggest that the reduced capability of alveolar macrophage phagocytosis for NTHi in COPD is due to the decrease in the extracellular expression of Siglec-1.

Although accumulating studies observed the defective phagocytosis of microbial pathogens including NTHi and S.pneumoniae by alveolar macrophages in COPD [4‐6], there are few reports that identified molecular mechanisms involved in the reduced phagocytic ability. Bewley et al. recently reported that up-regulation of the anti-apoptotic protein Mcl-1 and a failure to produce mitochondrial ROS in alveolar macrophages of COPD reduced intracellular killing of S.pneumoniae [34]. In addition Harvey et al. reported that Nrf2 activation improved the phagocytic ability for NTHi by increasing the scavenger receptor MARCO in alveolar macrophages derived from patients with COPD, though it has not been examined whether MARCO protein expression is down-regulated on the surface of alveolar macrophages of COPD compared to control smokers [35]. Thus our study firstly showed the possibility that an alteration of plasma membrane-bound phagocytic receptors might be a critical indicator for bacterial clearance of alveolar macrophages under inflammatory conditions.

The multivariable linear regression analysis revealed that the severity of pulmonary emphysema evaluated by the low attenuation area score on chest CT scans was an independent parameter associated with the decrease in Siglec-1 on alveolar macrophages. Although type 1 interferon was known to induce Siglec-1 protein expression in patients with systemic sclerosis [36] or with human immunodeficiency virus-1 [37], it has not been determined whether and how the Siglec-1 protein expression is down-regulated during pathological processes. Thus our finding provides new evidence of the reduced expression of Siglec-1 in COPD of which pathogenesis is involved in chronic inflammation related to excessive oxidative stress and would provide a rationale to investigate molecular basis of upstream signalling pathways negatively regulating Siglec-1 expression.

A large number of studies support the idea that Siglec-1-expressing tissue macrophages have critical roles in initiation of proper pro-inflammatory responses to maintain homeostasis in viral infection, cancer and autoimmune diseases [11, 17, 38]. This idea was further validated by specific depletion of Siglec-1-positive macrophages using Siglec-1-diphteria toxin receptor-transgenic mice [39, 40]. Taken together with these reports, our observation suggests that Siglec-1^dim-neg alveolar macrophages in COPD lungs might represent a dysfunctional macrophage for innate host defence.

There are some limitations in our study. First, the sample size of our flow-cytometric analysis was small with only mild and moderate COPD, because the availability of resected lung tissues, especially from patients with severe or very severe COPD, was limited. However it is noteworthy that Siglec-1 was decreased even in the early stages of COPD patients who had never experienced acute exacerbation. Second, we did not investigate a prospective cohort to determine whether patients with COPD who have lower expression of Siglec-1 on their alveolar macrophages exhibited higher bacterial colonisation or experienced more acute exacerbations. Further functional studies with prospective larger cohorts are needed to precisely define the roles of Siglec-1 on alveolar macrophages in innate host defence under pathologic conditions. Third, as we performed the in vitro phagocytosis assay with female control never smokers, we cannot exclude a possibility that gender and smoking status might affect this result. Fourth, because all patients in this study had primary lung cancers, the presence of tumour cells might suppress the expressions of phagocytic receptors and the ability of bacterial phagocytosis. Although we used peripheral lung tissues distant from tumor lesions, recent reports have indicated that cancer cells might have a crosstalk with distant cells via extracellular vesicles to inhibit anti-tumour immunity [41]. Fifth, small numbers of dendritic cells and monocytes might be included in the phagocytosis assay, since we did not exclude those cells using specific cell surface markers in flow cytometry. Finally, although we observed decreases in expression of FcγRI and MSR-1 in COPD macrophages compared to CNS, we did not determine whether Siglec-1^dim-neg macrophages also lost expression of FcγRI and MSR-1 or if decreased receptor expression in COPD alveolar macrophages had less specificity. It would be notable for a future study to evaluate multiple receptor expression of alveolar macrophages in diseased lungs.

We demonstrated that the extracellular expression of Siglec-1 was decreased on alveolar macrophages of COPD especially with pulmonary emphysema. Considering the involvement of Siglec-1 in NTHi phagocytosis by human alveolar macrophages, our data shed light on the importance of phagocytic receptors in alveolar macrophages in the pathogenesis of COPD.