A New Serum Macrophage Checkpoint Biomarker for Innate Immunotherapy: Soluble Signal-Regulatory Protein Alpha (sSIRPα)

Abstract

Background and Aims: The macrophage “don’t eat me” pathway CD47/SIRPα is a target for promising new immunotherapy. We hypothesized that a soluble variant of SIRPα is present in the blood and may function as a biomarker. Methods: Monocyte derived macrophages (MDMs) from human buffy-coats were stimulated into macrophage subtypes by LPS and IFN-γ (M1), IL-4 and IL-13 (M2a), IL-10 (M2c) and investigated using flow cytometry. Soluble SIRPα (sSIRPα) was measured in cell cultures and serum by Western blotting and an optimized ELISA. Serum samples were obtained from 120 healthy individuals and from 8 individuals challenged by an LPS injection. Results: All macrophage phenotypes expressed SIRPα by flowcytometry, and sSIRPα was present in all culture supernatants including unstimulated cells. M1 macrophages expressed the lowest level of SIRPαand released the highest level of sSIRPα (p < 0.05). In vivo, the serum level of sSIRPα increased significantly (p < 0.0001) after an LPS challenge in humans. The median concentration in healthy individuals was 28.7 µg/L (19.8–41.1, 95% reference interval), and 20.5 µg/L in an IFCC certified serum reference material. The protein was stable in serum for prolonged storage and repeated freeze/thawing. Conclusions: We demonstrate that sSIRPα is produced constitutively and the concentration increases upon macrophage activation both in vitro and in vivo. It is present in human serum where it may function as a biomarker for the activity of tumor-associated macrophages (TAMs), and for monitoring the effect of immunotherapy.

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“…On the other hand, CD47, the self-marker of most cells, inhibits phagocytosis by binding to signal regulatory protein α (SIRPα) of macrophages, transmitting the signal of “don't eat me”, thereby reducing the uptake of HMPB-Pt@MM by M0 phenotype [95] . However, the expression of SIRPα is reduced in M1 macrophages, thus alleviating “phagocytic inhibition” [96] . Because of these two reasons, HMPB-Pt@MM was taken up more by M1 macrophages and less by M0 macrophages.…”

Proinflammatory polarization strongly reduces human macrophage in vitro phagocytosis of tumor cells in response to CD47 blockade

“…After washing, plates were incubated with Avidin/HRP for 1 h. Control samples and standards ranging from 6.25 to 200 μg/L were included in each run. sSIRPα was measured in duplicate by an in‐house sandwich ELISA as previously described [23]. In brief, 96‐well microtiter plates were coated with capture antibody (polyclonal anti‐human SIRPα antibody (R&D systems, AF4546)) overnight.…”

“…M1 (IFN-γ+LPS) macrophage polarization strongly increased membrane expression of LILRB1 and Siglec-10 albeit in temporally distinct patterns. However, M1 polarization simultaneously induced extracellular domain shedding of SIRPα [22,23], measurable as slightly reduced membrane SIRPα levels and increased concentrations of soluble SIRPα (sSIRPα) in the culture supernatant after both 2 and 48 h of polarization with IFN-γ+LPS (Supporting information Fig. S6).…”

Comparison of culture media reveals that non‐essential amino acids strongly affect the phenotype of human monocyte‐derived macrophages

“…Culture supernatants were collected on day 6, centrifuged at 500 g/5 min/RT and stored at À80 C. Concentrations of soluble proteins were measured using in-house developed enzyme-linked immunosorbent assays (ELISA). Concentrations of sCD163 (samples diluted 1:10) [15], sCD206 (samples diluted 1:5) [16] and sSIRPα (samples diluted 1:10) [17] were measured as previously reported in detail, while a recently developed in-house ELISA assay was used for sLILRB1. In brief, Microtitre plates were coated with polyclonal anti-human LILRB1 antibody (R&D systems, AF2017) and incubated overnight.…”