S1P/HDAC1 Signaling Regulates Macrophage Polarization in Patients with Chronic Obstructive Pulmonary Disease

Throughout the world, chronic obstructive pulmonary disease (COPD) is the cause of substantial morbidity and mortality and represents a large public health burden. Previous studies reported that inammation is intimately involved in COPD, but the underlying mechanism remains unclear. As macrophage polarization was demonstrated to regulate inammation, we hypothesized that COPD alters macrophage polarization. To test this, we investigated in individuals with COPD the relationship between S1PR1 and HADC1 expression and macrophage inammation via ow cytometry, qRT-PCR, gene knockdown/overexpression, and western blot analysis. We found that macrophages from COPD subjects were polarized towards an M1 phenotype with increased S1PR1 and decreased HADC1experssion. S1PR1 also inhibited HADC1 expression. Thus, S1PR1/HDAC1 signaling regulates macrophage polarization. The results from this study provide insights into the pathogenesis of COPD and suggest possible therapeutic opportunities.


Introduction
Chronic obstructive pulmonary disease (COPD) results in signi cant morbidity and mortality [1]. COPD has been demonstrated to be the third leading cause of mortality worldwide [2]. The incidence of COPD in adults older than 40 years old is approximately 9-10% [1], and 5% of all deaths worldwide were due to this disease [3]. In China, the prevalence of COPD ranges from 1.20-8.87% [4]. Notably, the prevalence of COPD increases as smoking frequencies rise [5], while more than 90% of COPD cases stem from longterm cigarette smoking [6]. Additionally, COPD is closely associated with cardiovascular (CV) disease [7]. COPD is characterized by chronic bronchitis, destruction of small airways, and enlargement/disorganization of alveoli [8]. COPD is caused by multiple factors, such as infectious agents, including Haemophilus in uenzae, Moraxella catarrhine, and Streptococcus pneumonia [9]. Additionally, cellular-and antibody-mediated autoimmunity is thought to cause COPD, especially the in ammatory response presenting in the lower airways [10]. Increased cell senescence, which affects lung structure, and the types and numbers of in ammatory cells and broblasts, may also contribute to COPD [11]. However, the pathogenesis of COPD is not fully understood. Sphingosine 1-phosphate (S1P) plays a major role in the sphingolipid pathway [12]. Sphingolipid signaling regulates cell fate and immune responses [13]. S1P originates from ceramide inside cells, which is generated de novo or through the breakdown of membrane-resident sphingomyelin. S1P functions as a strong lipid mediator, regulating biological activities via its ve S1P cell membrane receptors (S1P1-5) [14]. S1P has been shown to regulate cell-cell and cell-matrix interactions via secretion of its proteincoupled receptors into the extracellular environment, thus closely governing cell migration, differentiation, and survival [15]. Dysregulated S1P signaling may contribute to disease, including atherosclerosis [14], in ammatory bowel [16] and Huntington's disease [17], and hepatocellular carcinoma [18]. S1P was linked to COPD, as several sphingolipid molecules modify in ammatory lung diseases and are therapeutic targets for treating lung injury and in ammation [19].
Macrophages play an important role in the pathogenesis of COPD. As the precursor of S1P, ceramide inhibits macrophage efferocytosis in COPD [20]. M1/M2 macrophage polarization is central to several in ammatory reactions [21]. Interleukin 10 (IL-10) and IL-12 are key factors in differentiating the M1 and M2 subtypes of macrophages [22]. HDACs were also linked to macrophage activity [23,24].
Herein, we examined expression of S1PR1 and HDAC1 in macrophages from controls and individuals with COPD, and by limiting and increasing signaling, assessed changes in cell activation.

Healthy volunteers and COPD patients
All healthy volunteers and patients enrolled in the study signed written informed consent. The study was approved by the ethics committee of Shenzhen University located in Shenzhen, China (reference NSSKH201908).

Isolation of PBMCs
PBMCs from healthy and COPD individuals were isolated using 9-mL Vacuette tubes with 3.8% sodium citrate (Greiner Bio-One, Kremsmünster, Austria). Blood samples were diluted with equal volumes of trisbuffered saline solution (TBSS, pH 7.5), inverted 10 times, and centrifuged at 1700 ´ g for 30 minutes at room temperature. The uppermost PBMC-containing buffy coat was removed, resuspended and

Flow cytometry
Flow cytometry was performed based on a standard protocol as published previously [25]. Brie y, PBMCs were dissociated with 0.25% trypsin (Sigma-Aldrich) and counted. Cells were washed twice using cold PBS, followed by adjusting the cell number to be 1 × 10 7 cells/mL. Cells were stained with 20 μL of  Table 1.  [29]. Brie y, when cell con uency researched 60% to 70%, the culture medium was removed and 1 mL of transfection medium was added. The ratio of plasmid to polyethylenimine was set at 1:3, which was then mixed, incubated at room temperature for 10 minutes, and then added into the wells of a 12-well plate containing the cells. Wells were mixed gently by swirling and then incubated for 3 hours in an incubator at 37°C with 5% CO 2 .
Three hours post-transfection, the old medium was removed, new medium was added, and the cells were incubated overnight in an incubator at 37°C with 5% CO 2 .

Statistical analysis
All data are expressed as the mean ± SEM. GraphPad Prism 6.0 software (San Diego, CA, USA) was used to calculate statistics. A Student's t test was used, and p < 0.05 was considered signi cant.

Results
GM-CSF macrophage stimulation and S1PR1 and HDAC1 expression in healthy and COPD patients GM-CSF was used for macrophage differentiation [26]. PBMCs from healthy and COPD individuals were treated with 100 ng/mL of GM-CSF for 14 days (Figure 1). The cells were then challenged with LPS (100 ng/mL) for 24 hours to generate macrophages. Interestingly, S1PR1 mRNA levels were signi cantly higher (Figure 2A, P < 0.001, n = 5), and HDAC1 mRNA levels were lower ( Figure 2B, P < 0.01, n = 5) in macrophages from COPD patients as compared to the healthy individuals. Paralleling changes in transcript, SIRP1 protein levels were increased ( Figure 2C and 2D, P < 0.01, n = 4) and HDAC1 levels decreased ( Figure 2E and 2F, P < 0.01, n = 5) in macrophages from individuals with COPD compared to controls.

Effect of S1PR1 and HDAC1 inhibition on macrophage polarization
It was not clear what role S1PR1 and HDAC1 had on macrophage polarization. Thus, macrophages were treated with shRNA to knockdown either S1PR1 (Figures 4A and 4B) and HDAC1 (Figures 4C and 4D). Knockdown of S1PR1 signi cantly increased IL-10 production in cells from both groups while suppression of HDCA1 decreased the production of IL-10 ( Figure 4E). Conversely, knockdown of S1PR1 signi cantly decreased IL-12 whereas knock down of HDAC1 increased production of IL-12 in macrophages from healthy and COPD individuals ( Figure 4F).
To further conform these data, macrophages from healthy controls were treated with FTY720. Similarly, limiting S1PR1 increased IL-10-positive cells, but decreased the number of IL-12-positive cells ( Figure 5B), as measured by ow cytometry. Additionally, inhibition of S1PR1 export with MK571 in healthy macrophages increased the number of IL-10-positive cells while decreasing IL-12-positive cells ( Figure   5C).
While S1PR1 and HDAC1 are known to regulate macrophages, studies regarding the effects of these genes on macrophage polarization are limited. Interestingly, a decrease in S1PR1 expression was found to increase the number of IL-10-positive cells and decrease the number of IL-12-positive cells, suggesting that S1PR1 supports an M1 phenotype. Further, S1Pr1-mediated suppression of HDAC1 increased the number of IL-12-positive cells and decreased the number of IL-10-positive cells, thus fostering an antiin ammatory signal. These data are also consistent with other reports that S1P inhibited HDAC1 activity through several mechanisms [42] [43]. Thus, the present study systematically studied the role of macrophage polarization in COPD patients. Importantly, the model of action involving macrophage polarization was dissected. Thus, this study provides insights into the pathogenesis of COPD and useful information for the development of novel therapies for the treatment of COPD. Figure 1 Time-dependent growth of GM-CSF-stimulated macrophages derived from healthy (A) and COPD individuals (B).

Figure 2
Expression of S1PR1 and HADC1 in healthy and COPD patients. mRNA expression of (A) S1PR1 (P < 0.001, n = 4) and (B) HDAC1 (P < 0.01, n = 4) in macrophages derived from COPD individuals as compared with healthy controls. Protein level of (C, D) S1PR1 (P < 0.01, n = 4) and (E, F) HADC1 (P < 0.01, n = 4) in macrophages derived from COPD individuals as compared with healthy controls.  The concentration of IL-12 in the culture medium of macrophages with S1PR1 and HADC1 knockdown (*P < 0.05, n = 4).