Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3

Abstract

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic lymphocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. Here, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163⁺CD206⁺ MΦ to PGN for 24h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the pro-efferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36 and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1 and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Keywords: ADAM17; Bacillus anthracis; MerTK; efferocytosis; peptidoglycan.

Figure 1:. Bacillus anthracis peptidoglycan (PGN) inhibits efferocytosis in the presence of human serum.

A) Secreted TNF and IL-10 (24hr-treated Mϕ, in various serum conditions) from 4 independent donors. B) Gating strategy for flow cytometric evaluation of efferocytosis C) Percentage of CD64+ Mϕ containing apoptotic eFluor670-labeled PMN from the indicated serum conditions in the presence or absence of 10ug/mL PGN from ≥5 independent donors analyzed by two-way ANOVA with Sidak’s multiple comparison. D) Alternative display of human serum condition data from Figure 1C to demonstrate hi huS has lower efferocytosis compared to the other human serum in untreated Mϕ.

Figure 2.. Human M2-like MΦ reduce efferocytic receptor expression in response to PGN.

(A, C). Representative histograms of receptor expression. (B, D) MFI of surface efferocytosis receptors from the indicated serum conditions from ≥3 independent donors analyzed by two-way ANOVA with Sidak’s multiple comparison.

Figure 3:. PGN-treated supernatants have increased levels of ADAM17 substrates

A) Concentrations of soluble receptors and cytokines from efferocytosis supernatants that are known to be cleaved by ADAM17. B) %CD64+eFluor670+ efferocytosis from 10 donors, PGN-treated values were used for the correlation analysis C) Pearson correlation and Linear Regression analysis between %efferocytosis and concentration of TNF. PGN values are from panel B. All analytes were measured from 24hr PGN-treated supernatants in the non-hi huS serum condition analyzed by paired t-tests.

Figure 4:. Pharmacological inhibition of ADAM17 partially restores surface receptor expression and efferocytic capacity.

A) Mean % efferocytosis (left) or an alternative display of the data showing the same donor tracked across each treatment group (right). B) TNF concentrations from donor supernatants. C) Level of receptor expression on cell surface (MFI). Data are from 11 independent donors after 16hr pre-treatment with 10μg/mL PGN in the presence of 10% non-hi huS serum alone or in combination with ADAM17 inhibitors. A, B and C are paired data from the same donors