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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Inflamm Res. 2022 Jan 12;71(3):321–330. doi: 10.1007/s00011-022-01539-5

MicroRNA-181c-5p modulates phagocytosis efficiency in bone marrow-derived macrophages

Sarojini Singh 1,*, John C Henderson 1,*, Mallikarjun Patil 1,*, Praveen Kumar Dubey 1, Shubham Dubey 1, Ramaswamy Kannappan 1, Jianyi Zhang 1, Prasanna Krishnamurthy 1,#
PMCID: PMC8919373  NIHMSID: NIHMS1786181  PMID: 35020000

Abstract

Objective and design

Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis.

Materials and methods

miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively.

Results

miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Further, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis.

Conclusion

Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.

Keywords: miRNAs, macrophage, phagocytosis, miR-181c-5p

Introduction

Phagocytosis, the clearance of pathogens, cell debris, and apoptotic cells by phagocytes, is critical for tissue homeostasis, wound healing, and immune defense [1–3]. Professional phagocytes such as macrophages recycle apoptotic and necrotic cells, and the importance of this activity is magnified during tissue injury [1, 3, 4]. This is due to the large number of cells that undergo apoptosis concurrently, requiring immediate clearance to minimize tissue damage [5, 6]. Phagocytosis acts as one of the early steps in the innate immune response, and the efficacy of cell clearance can affect the subsequent immune response, and ultimately the wound healing outcome [1, 7–10]. Effective macrophage phagocytosis depends on a complex signaling process involving phagocyte recruitment, apoptotic cell recognition and phagocytic engulfment, and phagosome digestion and processing; and the success of each signaling process is critical for the effective clearing of the apoptotic cells [4, 11, 12].

MicroRNAs (miRNAs) have been shown to regulate a number of biological processes and have been implicated in numerous disorders and diseases [13–16]. miRNAs are small non-coding RNAs that regulate several signaling pathways, including those regulating phagocytosis [17–19]. miRNAs have been widely identified to regulate various aspects of macrophage biology, including macrophage development, polarization and plasticity, as well as their functions [20, 21]. A plethora of studies have shown the critical functions of different miRNAs in macrophages and their effects on health and disease [18, 22, 23]. Moreover, activation of inflammatory pathway, respiratory burst, and phagocytic signaling cascade could also modulate the miRNA profile of phagocytic cells such as macrophages. Studies have shown that induction of miR-146a, miR-155, and other miRNAs are NF-κB-responsive and are upregulated in phagocytes by inflammatory stimuli [24, 25]. AKT1-mediated and reactive oxygen species-dependent regulation of miRNAs are also reported in macrophages [26][27].

The members of the miR-181 family (miR-181a/b/c/d) play crucial roles in tissue inflammation and immune response [28]. Among the mir-181 family of miRNAs, miR-181c has been shown to play a significant role in cancer through its ability to promote proliferation, cell invasion, and migration and is highly upregulated in many cancer types [29–31]. While this molecule has been well studied across a number of cancer pathologies, it has yet to be explored what role it plays in macrophage biology, more specifically, the phagocytosis process. Our initial miRNA screening showed that miR-181c-5p is significantly upregulated during macrophage phagocytosis. Therefore, in this study, we aimed to explore the role of miR-181c-5p in macrophage clearance of apoptotic cells and showed that miR-181c-5p overexpression promoted macrophage phagocytosis. Our findings provide a novel outlook of miR-181c-5p function in the macrophage phagocytosis process.

Materials and methods

Cell culture

Mouse macrophage cell line RAW 264.7 (ATCC, USA) and rat cardiomyoblast cell line H9c2 (ATCC, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, USA) with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml of penicillin, 100 μg/ml of streptomycin) in a humified incubator with 5% CO2.

For bone marrow-derived macrophage (BMDMs) isolation, C57BL/6J (~8–10 weeks old, Jackson Laboratory, ME, USA) mice were used. All experiments conform to protocols approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham (IACUC-UAB). BMDMs were isolated from the bone marrow of C57BL/6J mice, as described previously [32]. Isolated BMDMs were selected by culturing in macrophage-specific DMEM media supplemented with L929-conditioned media.

Microarray and bioinformatic analysis

RAW 264.7 cells were exposed to apoptotic H9c2 cells (apoptosis through UV exposure, 10 min) for 2 h. Total RNA was isolated from the control and apoptotic H9c2-treated macrophages and sent for microarray screening and bioinformatic analysis (LC Sciences, LLC, TX, USA). From the bioinformatic analysis, significant miRNAs were screened using DIANA Tools [33]. Following pathway prediction using the TargetScan database, miRNAs were screened by the following credentials: significant change due to the introduction of apoptotic cells, predicted activity in phagocytosis relevant pathways (e.g., phagocytosis, cytoskeletal remodeling, and endocytosis), and lack of previous studies in macrophage phagocytosis.

Cell transfection

hsa-miR-181c-5p mimic (mirVana™miRNA mimic, Cat#4464066, assay id: MC10181), hsa-miR-181c-5p inhibitor (mirVana™miRNA inhibitor, Cat#4464084, assay id: MH10181) and the respective nonspecific controls (mirVana™ miRNA mimic negative control, Cat#4464058; mirVana™ miRNA inhibitor negative control, Cat#4464076) (Ambion, Thermo Fisher Scientific, USA) were used for transfections. Briefly, BMDMs were seeded in the macrophage-specific medium 24 h before transfection. The miR-181c-5p mimic, inhibitor, and respective controls were transfected with Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. After 24 h, the medium was replaced with complete DMEM medium without L929 medium overnight. Transfection efficiency was confirmed with TaqMan™ qPCR. The cells from each group were either used for phagocytosis assay or harvested for gene expression and western blot analysis.

Fluorescent bead uptake assay

The uptake of fluorescent beads was optimized using RAW 264.7 macrophages and 1 μm particle size fluorescent beads (yellow-green fluorescence, Cat# L4655, Sigma-Aldrich, USA). Macrophages were labeled with PKH26 (red fluorescence, Cat# MINI26-1KT, Sigma-Aldrich, USA) according to the manufacturer’s instructions and were seeded in 4-well glass chamber slides for 24 h. Five microliters of the bead solution were opsonized by resuspending with 5 mL of 50% FBS/PBS (phosphate-buffered saline) solution and incubated at 37 °C for 30 min. After opsonization, beads were seeded at increasing cell to bead concentrations [No beads (control), 1:12, 1:24 1:72, 1:150] and incubated at 37 °C for 2 h. Following engulfment, cells were washed 3 times with PBS to remove unengulfed beads. For fluorescence microscopy, the cells were fixed with 4% paraformaldehyde, washed with PBS, and mounted for fluorescence microscopy (Olympus IX83 fluorescence microscope). After optimization, the same experiment was repeated with BMDMs with a 1:150 cell to bead ratio.

Flow cytometry

To evaluate the expression of miR-181c-5p between engulfing and non-engulfing BMDMs, fluorescence-activated cell sorting (FACS) was performed. For FACS analysis, PKH26-labeled BMDMs were incubated with yellow-green fluorescent beads (1BMDM:150 beads) for 2 h, washed with PBS, and analyzed by flow cytometry. Engulfing (FITC+/PKH26+) and non-engulfing (only PKH26+) BMDMs were sorted using FITC/PE channel sorting. The sorted cells were collected and immediately subjected to RNA isolation. The expression of miR-181c-5p was analyzed by TaqMan™ real-time PCR.

Phagocytosis assay

BMDMs were plated at 5×104 cells/well in 8-well glass slides. The macrophages were labeled with cell-permeant dye Calcein, AM (Cat# C1430, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions for 2 h. Apoptosis in H9c2 cells was induced by hydrogen peroxide (H2O2) treatment (Sigma-Aldrich, USA) for 4 h. The apoptotic cells were labeled with PKH26 (red fluorescence, Catalog-MINI26-1KT, Sigma-Aldrich, USA) according to the manufacturer’s instructions. Apoptotic H9c2 cells were overlaid on BMDMs (1:1 ratio of macrophage: apoptotic cells) and incubated for 2 h. After 2 h, cells were washed 4 times with ice-cold PBS to remove the unengulfed apoptotic cells. The cells were fixed with 4% paraformaldehyde, counterstained with DAPI (Thermo Fisher Scientific, USA), and mounted for fluorescence microscopy (Olympus IX83 fluorescence microscope). Phagocytosis was determined by counting cells containing engulfed red fluorescent apoptotic cells. A minimum of 300 macrophages was counted per well in triplicate. Data were represented as percent phagocytosis, i.e., the total number of cells with ingested apoptotic cells divided by the total number of macrophages counted times 100. In a separate phagocytosis experiment, BMDMs were stained with Flash Phalloidin™ Green 488 (Cat# 424201, BioLegend, USA) to observe macrophage cell morphology.

Phagocytosis-associated gene array

To evaluate the effect of miR-181c-5p overexpression on phagocytosis-related genes, BMDMs were transfected with either hsa-miR-181c-5p inhibitor (mirVana™ miRNA inhibitor, Cat# 4464084, assay id: MH10181) or the negative control (mirVana™ miRNA negative control, Cat# 4464076). Following this, apoptotic H9c2 cells were added to the BMDMs for 2 h. After 2 h, total RNA was extracted using an RNA extraction kit (Qiagen, USA) according to the manufacturer’s instructions. RNA was reverse transcribed using qScript cDNA SuperMix kit (Cat# 95048, Quantabio, USA). Following this, qPCR was performed using RT2 profiler PCR arrays (Cat# 330231, Qiagen, USA) to screen the genes involved in the phagocytosis pathway. Relative mRNA expression was normalized to the Beta-2-Microglobulin (B2M) gene. Gene expression was represented as fold change versus negative control.

Real-time qPCR analysis

To evaluate the expression of miR-181c-5p, total RNA was extracted from cells using a miRNeasy RNA extraction kit (Cat# 217004, Qiagen, USA) according to the manufacturer’s instructions. The RNA was reverse transcribed using TaqMan™Advanced miRNA cDNA Synthesis Kit (Cat# A28007, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed in a QuantStudio 3 system (Applied Biosystems, Thermo Fisher Scientific, USA) using TaqMan™ miRNA assays [mouse miR-181c-5p (Cat# A25576, assay id: mmu482604_mir) and U6 snRNA (Cat# 4427975, assay id: 001973)] and TaqMan™ fast advanced master mix (Cat# 4444964, Thermo Fisher Scientific, USA). miRNA-181c-5p expression was normalized to the U6 gene and a comparative CT method was used for relative quantification.

Western blotting

For cell lysate preparation, BMDMs transfected either with mimic control or miR-181c-5p mimic (in the presence or absence of apoptotic H9c2 cells) were lysed in RIPA buffer (Cat# J63324, Alfa Aesar) with protease and phosphatase inhibitor cocktail. Protein concentrations were determined by Bradford assay (Bio-Rad, USA). Equal amounts of proteins were denatured in 4× Laemmli buffer, separated on denaturing SDS-PAGE gel (4–20%), and then transferred to 0.2 μm PVDF membranes. The membranes were blocked with 5% bovine serum albumin (BSA) or non-fat milk powder (w/v) in TBS-T for 1 h at room temperature. The membranes were incubated with primary antibodies; N-WASP, WAVE-2 (actin nucleation and polymerization antibody sampler kit, Cat# 8606, Cell Signaling Technology, 1:1000 dilution for each antibody), and β-Tubulin (Proteintech, Cat# 66240-1-Ig, 1:10000) overnight at 4 °C, followed by HRP-conjugated secondary antibodies. The protein bands were developed by enhanced chemiluminescence (Pierce) detection system. Images of the blots were acquired on a ChemiDoc™ Touch Imaging System (Bio-Rad, USA), and densitometric analyses were performed using ImageJ (NIH) software.

Statistical analysis

Data are presented as mean ± SD. Statistical analyses was done using Prism (GraphPad software, CA, USA). An unpaired two-tailed t-test was performed between 2 groups to determine statistical significance. Probability (P) value of < 0.05 were considered a significant difference.

Results

Phagocytosis induces significant changes in macrophage miRNA profile

Our previous study showed that miRNAs play an important role in regulating macrophage efferocytosis [18]. To explore phagocytosis-induced changes on the macrophage miRNA profile, RAW 264.7 cells were incubated with apoptotic H9c2 cells and allowed to phagocytose. After 2 h, total RNA was isolated from RAW 264.7 macrophages, and microarray analysis was performed. The miRNA profiling showed differential expression of several miRNAs in control and phagocytosed cells (Fig. 1a, b and supplemental file 1). These miRNAs were further screened on the basis of their involvement in phagocytosis and cytoskeletal remodeling pathways, as described in the methods. After bioinformatics analysis and screening, miR-181c-5p was found to be a critical miRNA of interest, due to its robust increase following phagocytosis. The predicted involvement of the specific miRNA in a number of phagocytic pathways and lack of studies in macrophage phagocytosis makes it the potential target for our study. To validate the increased miR-181c-5p expression due to phagocytosis, we treated primary BMDMs with apoptotic H9c2 cells for 2 h and evaluated the expression of miR-181c-5p by TaqMan™ qPCR. The results consistently showed a significant increase in miR-181c-5p expression in macrophages upon the treatment of apoptotic cells (Fig. 1c).

Fig. 1. Phagocytosis induces significant changes in macrophage miRNA profile.

Fig. 1

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(a) Heat map showing alteration in microRNAs expression in RAW 264.7 cells incubated without (MФ alone) or with apoptotic H9c2 (MФ + dead cells) for 2 h. (b) Relative expression of differentially expressed miRNAs from the heat map with p<0.01. (c) TaqMan™ qPCR validation of miR-181c-5p expression in bone marrow-derived macrophages (BMDMs) alone (control) or treated with apoptotic H9c2 (control+dead cells) for 2 h. miR-181c-5p expression was normalized to U6 snRNA and values are shown as fold change. Data are represented as mean ± SD, n=4, *P < 0.05

Increased miR-181c-5p expression is correlated with phagocytosis

Our previous reports have shown that changes to the miRNA profile may be altered in different biological processes, including efferocytosis [18, 34]. To test whether the increase in miR-181c-5p was due to phagocytosis, a fluorescent bead uptake assay was optimized. Labeled latex beads were seeded with RAW 264.7 macrophages at increasing concentrations to determine effective seeding density for the assay (data not shown). Upon determining optimal seeding density, BMDMs were isolated, labeled with PKH26, and incubated with fluorescent beads (yellow-green fluorescence) to confirm phagocytosis (1 cell:150 beads) for 2 h. The fluorescence microscopy confirmed bead uptake in BMDMs (Fig. 2a). In a separate experiment, PKH26-labeled BMDMs were incubated with fluorescent beads for 2 h and were sorted into engulfing (PKH26+/FITC+) and non-engulfing (only PKH26+) BMDMs by flow cytometry (Fig. 2b). Total RNA was isolated from each group, and miR-181c-5p expression was evaluated. It was observed that miR-181c-5p was markedly upregulated in BMDMs that had phagocytosed the beads, compared to the cells that had not (Fig. 2c).

Fig. 2. Increased miR-181c-5p expression is correlated with phagocytosis.

Fig. 2

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(a) Representative fluorescence image of fluorescent bead uptake (1cell:150 beads) by PKH26-labeled BMDMs (scale bar: 50 μm). (b) Representative dot plot of fluorescence-activated cell sorting (FACS) illustrating PKH26-labeled BMDMs which have engulfed fluorescent latex beads (FITC+/PKH26+, labeled POS on the plot) and the BMDMs which have not engulfed latex beads (only PKH26+, labeled NEG on the plot). (c) Relative expression of miR-181c-5p in non-engulfing and engulfing macrophages following FACS assessed by TaqMan™ qPCR. miR-181c-5p expression was normalized to U6 snRNA and values are shown as fold change compared to non-engulfing BMDMs. Data are represented as mean ± SD, n=3

miR-181c-5p upregulation increases phagocytosis efficiency in vitro

Our previous work has shown that miRNAs have the potential to regulate many cellular functions [18, 34, 35]. To study the role of miR-181c-5p in macrophage phagocytosis, an overexpression study was performed. BMDMs were transfected with either miR-1815p mimic or miRNA mimic negative control (mimic control). Transfection efficiency was determined by qPCR (Fig. 3a). Following transfection, BMDMs and apoptotic H9c2 cells were fluorescently labeled with Calcein AM and PKH26, respectively, and incubated together for 2 h. Fluorescence microscopic analysis showed that overexpression of miR-181c-5p increased the phagocytosis efficiency of BMDMs as compared to untreated control and mimic control (Fig. 3b, c). In a separate experiment, BMDMs were also labeled with phalloidin to view cell morphology (Fig. 3d).

Fig. 3. miR-181c-5p upregulation increases phagocytosis efficiency of macrophages.

Fig. 3

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(a) Transfection efficiency in BMDMs transfected either with miR mimic negative control (mimic control) or miR-181c-5p mimic was assessed by TaqMan™ qPCR. miR-181c-5p expression was normalized to U6 snRNA and values are shown as 2−ΔCt. Data are represented as mean ± SD, n=5, **P < 0.01. (b) Representative fluorescence image of BMDMs phagocytosis transfected either with mimic control or miR-181c-5p mimic. BMDMs, apoptotic H9c2 cells, and nuclei were labeled with Calcein AM, PKH26, and DAPI, respectively (scale bar: 50 μm). (c) Quantification of phagocytosis efficiency calculated as percentage phagocytosis. Data are represented as mean ± SD, n=3. (d) Representative fluorescence image of BMDMs phagocytosis transfected either with mimic control or miR-181c-5p mimic. BMDMs, apoptotic H9c2 cells, and nuclei were labeled with phalloidin-green 488, PKH26, and DAPI, respectively (scale bar: 10 μm)

miR-181c-5p affects cytoskeletal dynamics during macrophage phagocytosis

To explore the mechanism behind miR-181c-5p’s interactions in the phagocytic pathway, a phagocytosis-associated gene array was employed to observe which genes were being affected (Fig. 4). The array revealed that many phagocytic genes were affected by the inhibition of miR-181c-5p, notably those involved in cytoskeletal remodeling and phagosome formation. Since a change in actin dynamics is a crucial event during phagocytosis, next, we evaluated the effect of miR-181c-5p on actin nucleation and polymerization. Overexpression of miR-181c-5p induced the expression of WAVE-2 in BMDMs. The upregulation of WAVE-2 was more prominent after incubation with apoptotic cells (Fig. 5a, b). This observation further affirms that miR-181c-5p might be involved in actin cytoskeleton rearrangement during macrophage phagocytosis. However, we did not observe any significant changes in N-WASP expression.

Fig. 4. miR-181c-5p regulates the expression of phagocytosis-related genes.

Fig. 4

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RT2 profiler PCR array of phagocytosis-related genes in the BMDMs transfected either with miR-181c-5p inhibitor or negative control followed by incubation with apoptotic H9c2 cells. Relative gene expression was normalized to B2M and values are shown as fold change. Data are represented as mean ± SD, n=2

Fig. 5. miR-181c-5p overexpression regulates molecules involved in actin nucleation and polymerization.

Fig. 5

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Representative western blots showing the protein expression of N-WASP and WAVE-2 in BMDMs transfected with either mimic control and miR-181c-5p mimic in the absence (a, left panel) or presence (b, left panel) of apoptotic H9c2 cells. Densitometric quantification of proteins for the same (a and b, right panel). Values are shown as fold change normalized to β-Tubulin. Data are represented as mean ± SD, n=3, **P < 0.01

Discussion

The innate immune system is a critical component of combating pathogens, wound healing, inflammation resolution, and maintaining homeostasis [36, 37]. As previous reports have shown, defective phagocytosis clearance of dead cells or defective efferocytosis can lead to adverse clinical outcomes and conditions, such as the development of autoimmunity and chronic inflammation [38, 39]. In our previous report, we showed that miRNAs play a critical role in macrophage efferocytosis, and rescuing miRNA expression under diseased conditions could also rescue macrophage function [40]. To further explore the changes in miRNAs signature and their role in macrophage phagocytosis, we performed an unbiased miRNA screening that revealed a number of miRNAs with modified expression during phagocytosis. Based on bioinformatics analysis and screening (described in the methods and results sections), miR-181c-5p was found to be a critical miRNA of interest, due to its robust increase following phagocytosis. Following the validation of the array results by qPCR, miR-181c-5p was used as a potential candidate for this study.

miR-181 family members (miR-181a, miR-181b, miR-181c, and miR-181d) play crucial roles in inflammation, endothelial cell activation, leukocyte biology, and immune response [28]. Moreover, miR-181c has pleiotropic function in different biological processes, including cell apoptosis [41, 42], mitochondrial function [28, 43], as well as inflammation and immune homeostasis [26, 28]. miR-181c-5p has also been reported to regulate bone formation [44], differentiation of human-induced pluripotent stem cells towards pancreatic lineage [45], and neuronal cell apoptosis and inflammation [46]. However, there is very limited information about the role of miR-181c-5p in macrophage biology. Since the expression of miR-181c-5p was upregulated significantly in our miRNA screening and there is no previous evidence, we were intrigued to further explore its involvement in macrophage phagocytosis. To investigate whether miR-181c-5p’s expression was due to phagocytosis specifically, a bead engulfment assay was performed to quantify the expression of miR-181c-5p in BMDMs that had specifically taken up the opsonized beads. We found that miR-181c-5p was upregulated in macrophages that had phagocytosed as opposed to macrophages that had not engulfed the beads, giving evidence to support that miR-181c-5p was indeed playing a role in the phagocytic process. Moreover, a gain-of-function study using miR-181c-5p mimic also exhibited increased phagocytosis efficiency in BMDMs incubated with apoptotic H9c2 cells. These observations presented further proof of concept that miR-181c-5p might be regulating macrophage phagocytosis.

To better understand the mechanism, we then performed a phagocytosis gene array to establish a guide to which part of the phagocytosis pathway miR-181c-5p was affecting. Inhibition of miR-181c-5p in phagocyting macrophages resulted in alteration in many phagocytic genes, notably those involved in cytoskeletal reorganization and phagosome formation. Interestingly, the DIANA-miRPath v2.0 [47] tool showed Fc gamma R-mediated phagocytosis as one of the miR-181c-5p target pathways. The phagocytosis process is complex and rearrangement of the actin cytoskeleton is an indispensable key event of this complex mechanism [48]. During phagocytosis, actin dynamics is regulated by activation of a myriad of molecules, such as small GTPases (Rac and Cdc42) and Wiskott-Aldrich syndrome protein family members (e.g., WASP and WAVE) that ultimately leads to recruitment and activation of Arp2/3 complex to actin filaments and actin polymerization [48]. Based on the results from the phagocytosis gene array and predicted involvement of miR-181c-5p in phagocytosis, we were interested to examine if miR-181c-5p is affecting the cytoskeletal dynamics in phagocytic macrophages. Intriguingly, miR-181c-5p overexpression showed upregulation of WAVE-2 protein in macrophages when fed with dead cells. These observations strongly suggest a potential involvement of miR-181c-5p in macrophage phagocytosis.

While this study makes a strong case for miR-181c-5p’s role in the regulation of macrophage phagocytosis, there are still some limitations that were beyond the scope of this study to address. Some may point out that miR-181c-5p is found to be upregulated in cardiac apoptosis and may influence its gene expression in phagocytosing macrophages [49]. While this is true, we feel the evidence given from the loss- and gain-of-function study performed specifically in macrophages gives credence to the idea that this miRNA is indeed playing a role in the macrophages. Also, in the current study, we only examined one aspect of miR-181c-5p in the phagocytosis process, i.e., actin polymerization. Therefore, further studies are needed to identify key targets of miR-181c-5p to elucidate the mechanistic role of this particular miRNA in the macrophage phagocytosis process. Also, it is not clear how phagocytosis might induce changes in miRNA profile. Previous literature has shown that in phagocytic cells, inflammatory response, oxidative stress, or changes in signaling cascade alters miRNA profile and the induced miRNAs are shown to be NF-kB-responsive or mediated through AKT1 [24, 26, 27]. Based on these reports, we speculate that the expression of miR-181c-5p might be regulated through NF-κB or AKT1. However, more studies are warranted to confirm these possibilities and to determine if miR-181c-5p is cell-specific.

In conclusion, we demonstrate the role of miRNA-181c-5p in macrophage phagocytosis. Overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages. Our findings suggest that miR-181c-5p could be an important regulator of cytoskeletal rearrangement during macrophage phagocytosis and further investigations are warranted to gain a comprehensive insight into miR-181c-5p’s role in macrophage biology.

Supplementary Material

1786181_Sup_S1

Acknowledgements

This work is supported, in part, by National Institutes of Health (NIH) grants HL116729 (to P.K.), HL137411 (to P.K. and J.Z.), American Heart Association Transformational Project Award 19TPA34850100 (to P.K.) and T32 Training Grant T32EB023872 (to J.H.).

Footnotes

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Conflict of interest

All the authors declare that they have no conflict of interest.

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Supplementary Materials

1786181_Sup_S1
NIHMS1786181-supplement-1786181_Sup_S1.xlsx (118.6KB, xlsx)

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