The Role of Siglec-F as a Regulator of Alveolar Macrophage Function and Adaptation

2019 dissertation for MRes. Alveolar macrophages are found in a truly unique microenvironment of the lung alveoli, where they are in close contact with the respiratory epithelium. This close contact with the epithelium is essential as it is how these highly inflammatory cells (cells that respond quickly to immune response as dysregulation of homeostasis exacerbates pathology) are regulated to prevent excessive inflammation, which otherwise would damage the lung, possibly leading to Asthma or COPD. Meaning that in the absence of pathology, the inflammatory response of alveolar macrophages are limited. Interaction with the lung occurs when macrophages bind to the epithelium through a specific receptor such as CD200R and possibly Siglec-F. Siglec-F is a lectin on the surface of macrophages that binds glycoconjugates containing sialic acid on the lung epithelium and on mucins, which is speculated to negatively regulate alveolar macrophages. Though the specific role of Siglec-F as a regulator of macrophage is not fully understood.
The aim of this project is to explore changes in gene expression and functional outcomes of signalling through Siglec-F. An exogenous system was used to stimulate this receptor using antibody crosslinking, which is then confirmed by analysing recruitment of SHP-1 in western blot. We also aim to measure differences in cytokine production using a multiplex kit which can simultaneously measure several different cytokines. Lastly, we aim to use RNA sequencing to analyse changes in gene expression. This data will then be used to propose functional outcomes of Siglec-F signalling; improving the understanding of the role of the receptor in negative regulation of alveolar macrophages.
The western blot results showed successful recruitment of SHP-1 and thus successful targeting of Siglec-F. We also observed a reduction in proinflammatory cytokines TNF-α, IL-6, CXCL1 and anti-inflammatory cytokine TGF-β1 when crosslinked with Siglec-F in the presence of a known strong stimulator of the innate immune response (LPS). This reduction of cytokines indicates a negative regulatory function of the Siglec-F receptor, though potential functional effector functions of Siglec-F is not understood. Looking at changes in expression we found interesting putative targets of the Siglec-F signalling pathway including immune related genes
such as TREM2, Axl, and IL-6 as well as other genes associated with RNA synthesis and the control of protein translation. This study will provide information about the effector functions of Siglec-F and creates new avenues for future study.