MicroRNA-34a Negatively Regulates Efferocytosis by Tissue Macrophages in Part via SIRT1

Abstract

Apoptotic cell (AC) clearance (efferocytosis) is an evolutionarily conserved process essential for immune health, particularly to maintain self-tolerance. Despite identification of many recognition receptors and intracellular signaling components of efferocytosis, its negative regulation remains incompletely understood and has not previously been known to involve microRNAs (miRs). In this article, we show that miR-34a (gene ID 407040), well recognized as a p53-dependent tumor suppressor, mediates coordinated negative regulation of efferocytosis by resident murine and human tissue macrophages (Mø). The miR-34a expression varied greatly between Mø from different tissues, correlating inversely with their capacity for AC uptake. Transient or genetic knockdown of miR-34a increased efferocytosis, whereas miR-34a overexpression decreased efferocytosis, without altering recognition of live, necrotic, or Ig-opsonized cells. The inhibitory effect of miR-34a was mediated both by reduced expression of Axl, a receptor tyrosine kinase known to recognize AC, and of the deacetylase silent information regulator T1, which had not previously been linked to efferocytosis by tissue Mø. Exposure to AC downregulated Mø miR-34a expression, resulting in a positive feedback loop that increased subsequent capacity to engulf AC. These findings demonstrate that miR-34a both specifically regulates and is regulated by efferocytosis. Given the ability of efferocytosis to polarize ingesting Mø uniquely and to reduce their host-defense functions, dynamic negative regulation by miR-34a provides one means of fine-tuning Mø behavior toward AC in specific tissue environments with differing potentials for microbial exposure.

Copyright © 2016 by The American Association of Immunologists, Inc.

Figures

Figure 1. miR-34a negatively regulates efferocytosis

A. AMø, glia, BMDMø and PMø were either incubated with AC in chamber slides for 1.5 h, then stained using H&E and assayed for efferocytosis (left-hand axis), or were assayed by quantitative real-time RT-PCR for expression of miR-34a relative to sno-142 (right-hand axis). Note inverse relationship between phagocytic index and miR-34a expression. Data are mean ± SEM of at least three replicates of each cell type from two independent experiments. B. Using RNAiMAX Lipofectamine, AMø, glia, BMDMø were transfected with control or miR-34a-specific antagomirs and PMø were transfected with control or miR-34a-specific mimics. AC engulfment was assessed by microscopy after 1.5 h. Data are mean ± SEM of at least three replicates of each cell type from two independent experiments; *, p

Figure 2. miR-34a negatively regulates efferocytosis by human AMø

AMø were harvested by bronchoalveolar lavage of healthy human never-smoker volunteers and transfected in vitro using control (scrambled) or miR-34a-specific antagomirs using RNAiMAX lipofectamine. Following transfection, AMø were either (A) processed to isolate total RNA, which was assayed by quantitative real-time PCR; or (B) exposed to AC for 90 min and then assayed for efferocytosis in chamber slides, which were H&E-stained and counted by microscopy. Data are mean ± SEM of individual subjects in independent experiments, A, n=3; B, n=4; *, p<0.05 by one-way ANOVA with Bonferroni post-hoc testing.

Figure 3. miR-34a negatively regulates binding to AC but not uptake of other targets

A–D. Using RNAiMAX Lipofectamine and chamber slides, murine AMø were transfected with control or miR-34a-specific antagomirs (24 h incubation) and murine PMø were transfected with control or miR-34a-specific mimics (48 h incubation). A. AC adhesion by transfected AMø and PMø was assessed by histology after 15 min. Data are mean ± SEM of three independent experiments. B. Phagocytosis by transfected AMø was assessed by microscopy after 1.5 h exposure of AMø to live thymocytes, necrotic thymocytes, or opsonized SRBC. Data are mean ± SEM of at least three independent experiments per target cell type. C, D. Phagocytosis by transfected PMø of pHrodo-labeled, Ig-opsonized-S. aureus following 1 h exposure, then release and analysis by flow cytometry. C. Representative histogram showing pHrodo staining. D. Uptake as measured by pHrodo MFI; data are mean ± SEM from n=3 mice assayed individually in each of two independent experiments, p<0.05 by one-way ANOVA with Bonferroni post-hoc testing.

Figure 4. miR-34a regulates Mø expression of Axl, but altered Axl expression is not required for regulation of efferocytosis

A, B. AMø from wt mice were transfected with control scrambled antagomirs (scrambled) or miR-34a-specific antagomirs (miR-34a KD) using RNAiMAX lipofectamine. Following knockdown, AMø were stained for surface expression of Axl and assayed by flow cytometry, gating AMø as CD45+ CD11c+ cells. A. Representative histograms; isotype, grey; untreated, dashed line; miR-34a+/− KD, solid line; scrambled antagomir-treated omitted for clarity. B. Data are expressed as fold change in MFI, relative to untreated AMø and are presented as mean ± SEM of as least four replicates in each of three independent experiments; ***, p

Figure 5. miR-34a negatively regulates Mø SIRT1 expression and SIRT1 enhances efferocytosis

A. Total RNA was harvested from AMø of wt mice and miR-34a+/− mice and SIRT1 mRNA expression was assessed by quantitative real-time RT-PCR, relative to GAPDH. B. Resident AMø from wt mice were treated in chamber-slides with the SIRT1-agonist resveratrol (10 μM for 24 h), exposed to AC and then efferocytosis was measured by microscopy. C–E. Resident PMø from wt mice were treated in chamber-slides with the SIRT1-antagonists (C) EX-527 (10 μM for 24 h) or (D, E) sirtinol (10 μM for 24 h), exposed to AC for 90 min, washed using a systematic protocol, stained using H&E and then efferocytosis was measured by microscopy. (F, G) Resident PMø from wt mice were treated in chamber-slides with the SIRT1-antagonist sirtinol (10 μM for 24 h), exposed to Ig-opsonized SRBC for 60 min, washed using a systematic protocol, stained using H&E and then FcγR-mediated uptake was measured by microscopy. Representative photomicrographs (D, F) are H&E-stained at 1000 X magnification. Data in all graphs are mean ± SEM from at least four replicates from two or more independent experiments. *, p

Figure 6. Efferocytosis down-regulates miR-34a, creating a positive-feedback loop for secondary engulfment

A. Resident AMø from wt mice were exposed to AC f for 2 h, then AC were removed by vigorous washing and AMø were incubated in LCM for a further 22 h before a second aliquot of AC were added and efferocytosis was assayed after 90 min using a microscopy assay of H&E stained slides. B. Resident AMø from wt mice were exposed to AC or LCM for 24 h, washed to remove unbound AC and then total RNA was harvested; expression of miR-34a, relative to sno-142, was assessed using quantitative real-time RT-PCR. C. Resident PMø from wt mice were exposed to AC twice, exactly as in panel A, except that in the second exposure, AC were TAMRA-labeled and efferocytosis was assayed after 60 min using flow cytometry. C. Data in all graphs are mean ± SEM from at least four replicates from two or more independent experiments. *, p