Multiple cell types can engulf and clear apoptotic cells from tissues in a process called efferocytosis. An integral function of the immune system, efferocytosis removes dead cells before they leak and spread toxins, bacteria, or proteolytic enzymes throughout the tissue. This process must be tightly regulated, because defects in efferocytosis can alter tissue homeostasis and contribute to diseases, such as systemic lupus erythematosus and other autoimmune disorders. In a new study published in The Journal of Immunology, a research team led by Dr. Jeffrey Curtis with the University of Michigan Health System investigated a novel role of microRNA miR-34a in regulating the efferocytotic activity of tissue resident macrophages, the most active cells engaged in removing cellular detritus. Manipulating miR-34a expression levels in macrophages in vitro led to inverse changes in efferocytosis by these cells in culture. These data suggest that manipulating tissue macrophage miR-34a expression may be a viable strategy for treating individuals with conditions that arise from abnormal efferocytosis.
MicroRNAs (miRs) are short, single-stranded RNA molecules that regulate gene expression primarily through post-transcriptional binding to target mRNA molecules. MicroRNA-34a and its family members have been implicated in many cancer types, and one target of miR-34a gene inactivation is the silent information regulator 1 (SIRT1). SIRT1 inhibition leads to p53 acetylation, changes in the cell cycle, and induction of apoptosis. In order to examine the relationship between miR-34a expression and apoptotic cell engulfment by macrophages, Dr. Curtis’s group began by isolating resident macrophages from alveoli, brain, bone marrow, and the peritoneum of C57BL/6J (000664) mice. Expression of miR-34a was measured by RT-PCR, and mRNA levels were compared to the macrophages’ efferocytotic activities in vitro. The ability of each macrophage type to ingest apoptotic cells was inversely related to the level of miR-34a expression level: for example, alveolar macrophages expressed the least miR-34a but had the highest efferocytotic activity. In contrast, peritoneal macrophages expressed the highest miR-34a levels but cleared the fewest apoptotic cells in culture. To assess whether miR-34a expression directly regulates efferocytosis, the researchers modulated miR-34a expression in macrophages in vitro by transiently transfecting them with miR antagomirs (anti-sense RNAs) and in vivo by genetic knockdown by crossing mice carrying a miR-34a conditional allele (STOCK Mir34atm1.2Aven/J (018545)) to mice expressing cre recombinase specifically in macrophages (B6.129P2-Lyz2tm1(cre)Ifo/J (004781)). In both experiments, reducing miR-34a expression increased macrophage efferocytosis in their in vitro assay. Efferocytosis also was reduced following transient transfection of miR-34a antagomirs in alveolar macrophages collected from healthy human volunteers, demonstrating that this mechanism is conserved in humans.
Next, the Curtis team sought to describe the mechanism through which miR-34a affects efferocytosis. Because SIRT1 is a known miR-34a target, they began by examining Sirt1 mRNA expression in alveolar macrophages in which miR-34a was knocked down using the Cre-lox strategy described above. Macrophages from these mice with only one functional miR-34a gene had increased Sirt1 expression, which was expected. Further, treating alveolar macrophages from wild-type mice with resveratrol, a SIRT1 agonist, increased efferocytosis in vitro. Lastly, Dr. Curtis’s group showed that miR-34a expression itself is inhibited in alveolar macrophages 24h after exposing them to apoptotic cells in vitro. The reduced miR-34a levels in turn stimulate secondary efferocytosis by the resident macrophages. Together, these results demonstrate that miR-34a regulates efferocytosis via a positive feedback loop that promotes secondary efferocytosis by resident macrophages following an initial encounter of apoptotic cells. The positive feedback is mediated by reduced miR-34a expression levels.
This study describes a novel role for a cancer-associated miR, miR-34a, in immunity by regulating efferocytosis by resident tissue macrophages. Understanding this mechanism may uncover novel therapeutic strategies for treating patients with conditions that are driven by defects in efferocytosis, such as some autoimmune disorders.