MicroRNA-21 Limits Uptake of Listeria monocytogenes by Macrophages to Reduce the Intracellular Niche and Control Infection

“Their expression of pattern recognition receptors (PRRs) (Takeuchi and Akira, 2010), both on the cell membrane and in the cytosol, allows them recognize potentially harmful bacteria.”

“…| www.frontiersin.org 7 May 2017 | Volume 7 | Article 201 expressed on the phagocyte cell surface and membranes of vesicular compartments (Janeway and Medzhitov, 2002; O’Neill et al., 2013) This results in the initiation of appropriate intracellular signaling pathways that trigger active…”

“MARCKS has recently been shown to be a target of miR-21 in epithelial cells, where its expression influences mucin secretion (Vazquez-Boland et al., 2001; Li et al., 2009; Lampe et al., 2013).”

“expressed on the phagocyte cell surface and membranes of vesicular compartments (Janeway and Medzhitov, 2002; O’Neill et al., 2013) This results in the initiation of appropriate intracellular signaling pathways that trigger active phagocytosis and also production of cytokines to alert nearby immune cells (Kaufmann and…”

“NO which is produced via the enzymatic activity of inducible nitric oxide synthase 2, has been shown to be an important mediator of immune responses to Listeria (MacMicking et al., 1995; Endres et al., 1997).”

“Macrophages are important effector cells of the innate immune system and represent the first line of defense against invading bacterial pathogens (Benoit et al., 2008).”

“The role of miRNAs in immunity has been an area of intense research in recent years, and many have been implicated in the regulation of immune cell function including the fine-tuning of PRR signaling (Baltimore et al., 2008; O’Neill et al., 2011; Quinn and O’Neill, 2011).”

“…and actin rearrangement including the RhoGTPase, RhoB, which has been reported to operate in coordination with Cdc42 (Allen and Aderem, 1995; Caron and Hall, 1998; Carballo et al., 1999; Corradin et al., 1999; Zhang et al., 2005; Quinn et al., 2009; Yang et al., 2013; Choi et al., 2015).”

“…cytokine data supports this model, providing evidence for the miR-21 being a negative regulator of pro-inflammatory responses by inducing anti-inflammatory IL-10, as well as supporting the pre-existing studies for miR-21’s negative regulation of TNF-α (Sheedy et al., 2010; Barnett et al., 2016).”

“…undergoes a series of maturation steps which involves fusion with endosomal vesicles and fission vesicles, moving through early, intermediate and late stages culminating in formation of the mature phagolysosome which has acquired the full bactericidal repertoire (Weiss and Schaible, 2015).”

“MiR-21 is one of the most highly expressed miRNAs in many mammalian cell types (Krichevsky and Gabriely, 2009).”

“The role of miRNAs in immunity has been an area of intense research in recent years, and many have been implicated in the regulation of immune cell function including the fine-tuning of PRR signaling (Baltimore et al., 2008; O’Neill et al., 2011; Quinn and O’Neill, 2011).”

“A study of asthma showed that miR-21 negatively regulates immune responses in dendritic cells, by controlling the production of pro-inflammatory IL12 (Lu et al., 2009, 2011).”

“The production of NO during the oxidative burst which accompanies engulfment is a key antimicrobial mechanism of phagocytic cells, and has been shown to be important for the clearance of L. monocytogenes (Shiloh et al., 1999).”

“It was shown to regulate expression of the anti-inflammatory cytokine IL-10 in macrophages in response to bacterial LPS, by targeting PDCD4, a negative regulator of IL-10 translation (Sheedy et al., 2010; Sheedy, 2015).”

“MARCKS has recently been shown to be a target of miR-21 in epithelial cells, where its expression influences mucin secretion (Vazquez-Boland et al., 2001; Li et al., 2009; Lampe et al., 2013).”

“MiRNA are short, non-coding RNAs that have been shown to affect numerous cellular processes in a posttranscriptional manner (Bartel, 2004; He and Hannon, 2004; Almeida et al., 2011).”

“A study of asthma showed that miR-21 negatively regulates immune responses in dendritic cells, by controlling the production of pro-inflammatory IL12 (Lu et al., 2009, 2011).”

“In addition, the high bacterial burden present in our system may lead to an overwhelming cytokine signal, as demonstrated by the very high levels of the key L. monocytogenes cytokine IL-6 observed in response to infection (Dalrymple et al., 1995), which may hide potential differences.”

“The most well characterized phagocytic receptors on macrophages include FcγRs and complement receptor 3 (CR3), which bind to immunolglobulin G (IgG)-opsonised particles and complement-coated particles respectively (Kerrigan and Brown, 2009).”

“In addition, miR-21 has also been implicated in positively regulating the phenomenon of efferocytosis whereby activated macrophages alter their behavior to take up dying cells and prevent further inflammation (Das et al., 2014).”

“…involved in phagocytosis and actin rearrangement including the RhoGTPase, RhoB, which has been reported to operate in coordination with Cdc42 (Allen and Aderem, 1995; Caron and Hall, 1998; Carballo et al., 1999; Corradin et al., 1999; Zhang et al., 2005; Quinn et al., 2009; Yang et al.,…”

“The role of miRNAs in immunity has been an area of intense research in recent years, and many have been implicated in the regulation of immune cell function including the fine-tuning of PRR signaling (Baltimore et al., 2008; O’Neill et al., 2011; Quinn and O’Neill, 2011).”

“Key mediators of actin rearrangements are the RhoGTPases, which have been shown to be important for the initial formation of the phagocytic cup, which engulfs the invading pathogen (Rougerie et al., 2013).”

“MiR21 has previously been shown to be induced in response to bacterial LPS in macrophages while it is induced in monocytes infected with Mycobacterium leprae (Sheedy et al., 2010; Liu et al., 2012).”

“…and membranes of vesicular compartments (Janeway and Medzhitov, 2002; O’Neill et al., 2013) This results in the initiation of appropriate intracellular signaling pathways that trigger active phagocytosis and also production of cytokines to alert nearby immune cells (Kaufmann and Dorhoi, 2016).”

“Although the role of miR-21 in the host response to bacterial pathogens is relatively unexplored, this implies a potentially important role for miR-21 in the control of infection (Wang et al., 2015).”

“…and actin rearrangement including the RhoGTPase, RhoB, which has been reported to operate in coordination with Cdc42 (Allen and Aderem, 1995; Caron and Hall, 1998; Carballo et al., 1999; Corradin et al., 1999; Zhang et al., 2005; Quinn et al., 2009; Yang et al., 2013; Choi et al., 2015).”

“In particular, this increased MARCKS expression is found in areas of the cell where actin filaments associate with the plasma membrane, and its expression is associated with regulation of cell motility (Myat et al., 1997; Carballo et al., 1999).”

“monocytogenes uses listeriolysin (Hly), a thiol-activated cholesterol-dependent cytolysin to form pores in the membrane of the phagosome allowing escape into the cytosol (Singh et al., 2008).”

“NO which is produced via the enzymatic activity of inducible nitric oxide synthase 2, has been shown to be an important mediator of immune responses to Listeria (MacMicking et al., 1995; Endres et al., 1997).”

“However, dextran uptake is not necessarily a phagocytic process (Pustylnikov et al., 2014) and so we sought to confirm using a phagocytosis-specific assay.”

“We next analyzed another target of miR-21, Ras homolog gene family member B (RhoB), which has previously been reported to influence cytoskeletal changes during phagocytosis (Zhang et al., 2005; Quinn et al., 2009).”

“It would be interesting to assess the role of ROS in this system, as miR-21 has been demonstrated to impact generation of these antibacterial mediators in cancer settings (Jiang et al., 2014; Guo et al., 2015).”

“MiRNA are short, non-coding RNAs that have been shown to affect numerous cellular processes in a posttranscriptional manner (Bartel, 2004; He and Hannon, 2004; Almeida et al., 2011).”

“It would be interesting to assess the role of ROS in this system, as miR-21 has been demonstrated to impact generation of these antibacterial mediators in cancer settings (Jiang et al., 2014; Guo et al., 2015).”

“This failure of translation of ligand effect to physiological settings has been seen in other studies and points to a complexity in the role of the multi-target miR-21 in infectious settings (Barnett et al., 2016).”

“The ability of Listeria to establish an intracellular niche and evade immune surveillance typifies the struggle between infectious agents and the host immunity and is critical to the outcome of infection (Corr and O’Neill, 2009).”

“…induces actin tails through expression of an actin polymerizing protein, ActA that facilitate its propulsion through the cell cytosol toward the cell membrane, where it forms protrusions into neighboring cells allowing its internalization and facilitating cell-cell (Williams et al., 2012).”

“Macrophages have been shown to express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein (Carballo et al., 1999).”

“However, as with RhoB, further work is required to fully elucidate what role MARCKS might play in this system, particularly as it has been demonstrated that MARCKS and MacMARCKS deletion does not significantly impact phagocytosis in vivo or in vitro (Underhill et al., 1998).”

“…and actin rearrangement including the RhoGTPase, RhoB, which has been reported to operate in coordination with Cdc42 (Allen and Aderem, 1995; Caron and Hall, 1998; Carballo et al., 1999; Corradin et al., 1999; Zhang et al., 2005; Quinn et al., 2009; Yang et al., 2013; Choi et al., 2015).”

“…and actin rearrangement including the RhoGTPase, RhoB, which has been reported to operate in coordination with Cdc42 (Allen and Aderem, 1995; Caron and Hall, 1998; Carballo et al., 1999; Corradin et al., 1999; Zhang et al., 2005; Quinn et al., 2009; Yang et al., 2013; Choi et al., 2015).”

“MARCKS has recently been shown to be a target of miR-21 in epithelial cells, where its expression influences mucin secretion (Vazquez-Boland et al., 2001; Li et al., 2009; Lampe et al., 2013).”

“Following successful deletion, mice were back-crossed further to obtain a full body MiR-21−/− line described by (Johnston et al., 2017).”

“Induction of miR‐21 in macrophages infected by L. monocytogenes was shown to repress the actin mod- ulating proteins RhoB and MARCKS, decreasing bacterial phagocytosis during infection (Johnston et al., 2017).”

“Listeria monocytogenes ↑ miR-21 ↓ MARCKS and RhoB The pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) are downregulated to hinder pathogen internalization [37]”

“MiR-21 takes part in suppressing uptake by downregulating myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB); however, whether this regulation is by direct targeting, remains unclear [119].”

“For example, miR-21 expression is enhanced in murine bone marrow-derived M8 (BMDM) following infection with Listeria monocytogenes and BMDM of miR-21−/− mice show enhanced uptake of L. monocytogenes, FITC-labeled E. coli bioparticles, and FITC-dextran (38).”

“Negative control cells were incubated in FITC-dextran or FITC-zymosan solution at 4 ◦C (for live cells endocytosis and phagocytosis block) [70].”

“42 Hsa-miR-130a is relevant in the colonization of intestinal mucosa in Crohn’s disease patients, facilitating the intracellular replication of adherent–invasive Escherichia coli.”

“Increased MARCKS expression leads to increased uptake of Listeria monocytogenes, but there is no difference in either Fc- or CR-mediated phagocytic efficiency between MARCKS deficient cells and wild-type cells (Carballo et al., 1999; Johnston et al., 2017).”

“For example, miR-146, miR-155, miR-125, let-7, and miR21 are certainly involved in immune responses upon bacterial infection, such as with Mycobacterium tuberculosis [17,18], Helicobacter pylori [19,20], and Listeria monocytogenes [21].”

“Macrophages (Mø) are key effector cells of the innate immune system and constitute the first line of defense against bacterial invasion [21].”

“monocytogenes [8], Mycobacterium tuberculosis [26], and LPS treatment [27] of macrophages.”

“It was reported that microRNA-21, which has an anti-inflammatory function, limits uptake of L. monocytogenes cells by macrophages (Johnston et al., 2017).”

“Different bacterial infections are associated with upregulation of miR-21 in both in vitro and in vivo settings (Kelly et al. 2025; Johnston et al. 2017; Hackett et al. 2020; P. T. Liu et al. 2012).”

“Interestingly, a follow-up investigation found that miR-21 regulated phagocytosis to control L. monocytogenes infection via weakening the intracellular niche (Johnston et al., 2017).”