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 contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, 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 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic 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.

FIGURE 1.

B. anthracis PGN inhibits efferocytosis in the presence of human serum. (A) Secreted TNF and IL-10 (24 h–treated MФ in various serum conditions) from four independent donors. (B) Gating strategy for flow cytometric evaluation of efferocytosis. Gating strategy: After gating on MΦ and excluding dead cells and debris using scatter properties (forward scatter [FSC], side scatter [SSC]), MΦ with CD66b⁺ surface-bound apoptotic PMNs are excluded. Next, live MΦ are gated as CD11b⁺ and Zombie Aqua viability dye negative. Finally, we gate on CD64⁺ MΦ and measure the percentage of intracellular apoptotic PMNs present (CD64⁺ eFluor 670⁺). (C) Percentage of CD64⁺ MФ containing apoptotic eFluor 670–labeled PMNs from the indicated serum conditions in the presence or absence of 10 μg/ml PGN from ≥5 independent donors analyzed by two-way ANOVA with Sidak’s multiple comparison test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 2.

Human M2-like MΦ reduce efferocytic receptor expression in response to PGN. (A and C) Representative histograms of receptor expression. (B and D) Mean fluorescence intensity (MFI) of surface efferocytosis receptors from the indicated serum conditions from three or more independent donors analyzed by two-way ANOVA with Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, ****p < 0.0001.

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) Percentage CD64⁺ eFluor 670⁺ efferocytosis from 10 donors. PGN-treated values were used for the correlation analysis. (C) Pearson correlation and linear regression analysis between percentage efferocytosis and concentration of TNF. PGN values are from (B). All analytes were measured from 24-h PGN-treated supernatants in the non-hihuS serum condition and analyzed by paired t tests. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 4.

Pharmacological inhibition of ADAM17 partially restores surface receptor expression and efferocytic capacity. (A) Mean percentage 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 (mean fluorescence intensity [MFI]). Data are from 11 independent donors after 16-h pretreatment with 10 µg/ml PGN in the presence of 10% non-hihuS serum alone or in combination with ADAM17 inhibitors. (A), (B), and (C) are paired data from the same donors. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.