Helene Stern

Abstract

Macrophages (Mφs) exist on a phenotypic spectrum characterised by the polar extremes of two subsets, pro-inflammatory M1s and regulatory M2s. Both subsets express signal regulatory protein alpha (SIRPα), receptor to CD47. Characterised as a “don’t eat me” signal, SIRPα-CD47 ligation negatively regulates Mφ phagocytic activity. This study developed a co-culture system between CD47+ HL-60 and K562 cells, and SIRPα+ Mφ subsets in order to investigate whether the regulatory effect of SIRPα-CD47 ligation extends to negative regulation of TLR-mediated TNFα production, and how this differs between Mφ subsets and under various TNFα-inducing stimuli. SIRPα and CD47 expression in THP-1-derived M1- and M2-like Mφs, HL-60 pro-myelocytes and K562 lymphoblasts was measured using flow cytometry. THP-1 derived Mφs were co-cultured with either HL-60 or K562 cells. Alone and in co-culture, Mφs were stimulated with either Staphylococcus aureus LTA, Escherichia coli K12 LPS or Porphyromonas gingivalis LPS. TNFα production was measured by ELISA. M2-like Mφs were found to express higher levels of SIRPα than M1-like Mφs, and K562 cells expressed greater CD47 than HL-60 cells. In Mφ monoculture, all stimulation induced a TNFα response in both subsets, with M1s the higher TNFα producers in the case of K12 LPS and SA LTA stimulation, but no difference was present between subsets when stimulated with PG LPS. In all LPS co-culture conditions induced TNFα production was reduced compared to monoculture, with the higher SIRPα expressing M2 subset experiencing more suppression. The greatest suppressive effect was present when Mφs were stimulated with K12 LPS, compared to PG LPS. Co-culture did not inhibit SA LTA-induced TNFα production. When Mφs were co-cultured with the higher CD47-expressing K562 cells, a greater suppression of LPS-induced TNFα response was present than when Mφs were co-cultured with the lower CD47-expressing HL-60 cells. These data indicate a role of SIRPα-CD47 ligation in the negative regulation of TLR4-mediated macrophage pro-inflammatory response, with TLR2-mediated TNFα production unaffected by co-culture. These findings identify SIRPα as a potential therapeutic target for TNFα-mediated pathologies of uncontrolled inflammation including Crohn’s Disease and Rheumatoid Arthritis, presenting a novel mechanism for inducing functional shift from pro-inflammatory M1 towards regulatory M2 Mφ phenotype.

Keywords

Immunology, negative regulation, SIRPα, Homeostasis, Inflammation, CD47

Document Type

Thesis

Publication Date

2021

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