Siglec-5和Siglec-14受體對於巨噬細胞極化的影響

Abstract

巨噬細胞是具有高度可塑性的免疫細胞，會因為其遇到的微環境而被極化為M1或M2巨噬細胞，對於維持生理功能的恆定性相當重要。Siglec-5和Siglec-14為主要表現於巨噬細胞上的配對型受體，兩者在配體結合位具有極高的相似性，能在相同配體的刺激下分別經由本身的細胞內ITIM motif (Siglec-5)或是結合具有ITAM motif的DAP12 (Siglec-14)來傳遞相反的訊息。此外，腫瘤細胞可藉由其唾液酸修飾蛋白去鍵結巨噬細胞上的Siglecs受體來調控巨噬細胞極化成腫瘤相關巨噬細胞的能力，因此，本篇論文主要想探討巨噬細胞上的Siglec-5/14配對型受體在巨噬細胞分別被極化為M1、M2或是腫瘤相關巨噬細胞時所扮演的角色。我們會先利用LPS/IFN-γ和IL-4/IL-13來建立巨噬細胞極化的模式，隨後會將巨噬細胞培養在腫瘤細胞培養液中來研究Siglec-5/14是否可以調控腫瘤相關巨噬細胞形成。首先，經由比較發炎性激素的產生，我們知道表現Siglec-5/14與否並不影響THP-1細胞在LPS/IFN-γ刺激下形成M1巨噬細胞的能力。但表現Siglec-14的THP-1細胞在IL-4/IL-13的刺激能夠表現比較多的M2指標，如CCL18、CCL22、IL-1RA、CD36和CD204，以及表現較高量可促使M2巨噬細胞極化的轉錄因子，如PPAR-γ和LXR-α。我們發現，實驗所用到的三種大腸癌細胞(SW480、SW620、HT29)培養液都能夠促進THP-1細胞形成腫瘤相關巨噬細胞，而肺癌細胞(CL1-0、CL1-5)和乳癌細胞(BT549)則無法極化THP-1成為腫瘤相關巨噬細胞。在大腸癌細胞培養液的處理下，與腫瘤相關巨噬細胞有關的指標會因THP-1細胞表現Siglec-5或是Siglec-14而有所不同。Siglec-14/THP-1細胞能夠產生出較多的CCL18、CCL22、IL-1RA、CD36、CD204和IDO，而Siglec-5/THP-1細胞會產生出較多的IL-10和MMP-9。經由分析多種轉錄因子的表現或是活性，我們認為Siglec-14可能可以在大腸癌細胞培養液的處理下經由促進STAT3的活化和LXR-α的表現來增加TAM指標的表達。總結來說，我們發現在巨噬細胞上的Siglec-5和Siglec-14受體的確能在刺激下影響巨噬細胞的極化，但目前仍不明瞭兩者會透過什麼樣的機制來進行調控。

Macrophages are highly plastic immune cells which can be further polarized into M1 or M2 macrophages in response to their surrounding microenvironment, and they play a critical role in maintaining the physiological homeostasis of the body. Siglec-5 and Siglec-14 are paired receptors primarily expresssing on macrophages and have the same sequence of amino acid in their first two N-terminal domains which are critical for ligand binding. However, Siglec-5 delivers inhibitory signals through its intracellular ITIM motif while Siglec-14 transduces activating signaling through the coupled ITAM-containing adaptor, DAP12, upon ligand engagement. In addition, sialylated glycoproteins derived from tumor cells have been shown to target various Siglec receptors to modulate macrophage polarizing into tumor-associated macrophages (TAMs) which can generate an immunosuppressive and pro-tumoral environment to assist cancer progression. In this study, we aim to investigate the role of Siglec-5 and Siglec-14 in macrophage polarization in response to different environmental cues. We first tried to establish a THP-1 macrophage polarization system driven by LPS/IFN-γ and IL-4/IL-13 stimulation, and a successful polarizing stimulation was proved by examing the expression of classical markers often used to classify the M1 and M2 population. Next, expression of M2/TAM markers of the THP-1 macrophages cultured with the tumor conditional medium were studied to explore the impact of the paired Siglec receptors, Siglec-5 and Siglec-14, in TAM programming. Comparable expression of M1 macrophage markers were observed in the Siglec-5/THP-1 and Siglec-14/THP-1 cells upon LPS/IFN-γ treatment, suggesting that exogenous expression of Siglec-5 and Siglec-14 on THP-1 cells did not affect M1 macrophage polarization. Notably, increased expression of M2-specific markers, CCL18, CCL22, IL-1RA, CD36 and CD204, as well as transcriptional factor involved in M2 polarization, PPAR-γ and LXR-α, were detected in Siglec-14-expressing THP-1 cells upon IL-4/IL-13 treatment. Moreover, we found that conditional mediums (CMs) derived from all the tested colorectal cancer cells (SW480, SW620 and HT29) successfully induce THP-1 macrophage differentiating into TAMs, while CM derived from lung cnacer cells (CL1-0 and CL1-5) and breast cancer cells (BT549) failed to achieve the same effects in our experimental condition. Importantly, Siglec-5 and Siglec-14 differentially regulated the TAM-related markers expression of the THP-1 macrophages stimulated with colon cancer CMs. Siglec-14/THP-1 cells expressed more CCL18, CCL22, IL-1RA, CD36, CD204 and IDO, while Siglec-5/THP-1 cells generated more IL-10 and MMP-9. Siglec-14 may induce the activation of STAT3 and the transcription of LXR-α to facilitate TAM formation upon colon cancer CM treatment. In conclusion, our results demonstrate that Siglec-5 and Siglec-14 can modulate the polarization of macrophages in response to IL-4/IL-13 and colon cancer CM, but further studies are required to elucidate the involved mechanisms.