miR-185-5p Regulates Inflammation and Phagocytosis through CDC42/JNK Pathway in Macrophages

Macrophage activation is an essential component of systemic chronic inflammation and chronic inflammatory diseases. Emerging evidence implicates miR-185-5p in chronic inflammation diseases. However, the regulatory role of miR-185-5p in macrophage pro-inflammatory activation has not been studied previously. Here, we identified that miR-185-5p was one of the top genes and effectively downregulated in two macrophage miRNA expression datasets from GEO. Under LPS stress, miR-185-5p overexpression reduced pro-inflammatory cytokine expression, suppressed phagocytosis in RAW264.7 macrophage. miR-185-5p inhibitors augmented pro-inflammatory effects of LPS in macrophage. Mechanically, miR-185-5p sponged and negatively regulated the protein expression of CDC42. Ablation of CDC42 with selective CDC42 inhibitor CASIN reversed the pro-inflammatory effect of miR-185-5p inhibitors through inhibiting MAPK/JNK pathways. Collectively, these data demonstrate that miR-185-5p exhibited anti-inflammatory functions in LPS-induced RAW264.7 macrophages at least partially through CDC42/JNK pathways. Our findings yield insights into the understanding of miR-185-5p-regulated network in macrophages inflammation, which is beneficial for exploring miRNA-protein interaction in atherosclerotic inflammation.


Introduction
Atherosclerosis is a chronic inflammation disease, which is characterized by the development of atherosclerotic plaques in the vascular wall [1]. Atherosclerosis-related cardiovascular disease has become a leading cause of death worldwide [2]. Pro-inflammatory macrophages largely reside in intima and plaque shoulder of both mouse and human atherosclerotic plaques based on single-cell RNA-Seq findings [3]. Macrophage infiltration leads to non-resolving inflammatory condition [4]. In advanced atherosclerotic lesions, defective phagocytosis of apoptotic macrophages and incomplete clearance of cell debris amplify inflammation, which increases the risk of plaque rupture [5]. Monocytes and macrophages are hyper-inflammatory before recruitment to plaques in patients with atherosclerosis. In a human monocyte metabolism study, monocytes isolated from individuals with atherosclerosis produce higher cytokine than do monocytes from healthy individuals after stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) [6,7]. It indicates that the modulation of aberrant macrophage inflammatory responses is critical target in atherosclerosis.

RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA of cells was extracted by using TRIzol Reagent (Invitrogen, Gaithersburg, MD, USA). The cDNA synthesis kit (Takara, Kyoto, Japan) was used for reverse transcription of mRNA. The miRNA 1st Strand cDNA Synthesis (by stem-loop) Kit (Vazyme, Nanjing, China) was used to reverse transcribe miRNA. RT-qPCR was performed using the ABI QuantStudio 12K Flex Real-time PCR System (Life Technologies, Gaithersburg, MD, USA). TB Green ® Premix Ex Taq™ (Takara, Kyoto, Japan) was used for mRNA RT-qPCR, and miRNA Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for miRNA RT-qPCR. Relative gene expression was determined using the 2 -∆∆Ct cycle threshold method [28]. The mRNAs were normalized to endogenous control gene Actb, U6 was used as the internal control for miRNAs. The sequences of primers used are described in Table 2. Table 2. List of sequences used in the study.

ELISA Assays
After stimulating cells with 100 ng/mL LPS for 24 h, cell culture supernatants were centrifuged at 300× g for 10 min to remove cell debris. ELISA kits were used for mouse IL6 (Multi Sciences, Hangzhou, China) and mouse IL-1β (Multi Sciences, Hangzhou, China) detections according to the manufacturer's protocols. Optical density (OD) values at 450 nm and 570 nm were detected by a microplate reader (Bio-Tek, Winooski, VT, USA). Results were expressed as pg/mL.

Phagocytosis Assays
Phagocytosis was measured by Vybrant Phagocytosis Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) [29]. Cells were plated in black 96-well plates and incubated with FITC-labeled E. coli BioParticles for 2 h at 37 • C, then immediately incubated with trypan blue for 1 min at room temperature. Subsequently, cells were fixed with paraformaldehyde for 15 min and stained with DAPI. Cells were imaged using a fluorescence microscope Nikon Eclipse Ti-E (Nikon, Tokyo, Japan). The mean fluorescence intensity was analyzed by ImageJ software.

Statistical Analyses
Student's t test was used to analyze the statistical differences between two groups. Values were presented as means ± standard deviation (SD). Scatter histogram was performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Comparisons between multiple groups were evaluated by one-way ANOVA. Western blot quantification and fluorescence intensity quantification were analyzed and determined by ImageJ. p value < 0.05 was regarded as statistically significant.

Identification of the Candidate DEM
Two expression profiles (GSE87718 and GSE143845) were selected from the GEO database [26,27]. With the threshold of p value < 0.05 and |fold change (FC)| > 1, 129 DEMs (3 upregulated and 126 downregulated) were identified in GSE87718 ( Figure 1A,B). Total of 34 DEMs (14 upregulated and 20 downregulated) were identified in GSE143845 after screening with the threshold of an adjusted p value < 0.01 and |log FC| > 2 ( Figure 1C,D). Detailed information of the GSE87718 and GSE143845 datasets is shown in Supplementary Figure S1A-D. The Venn diagram indicated seven overlapping DEMs in the two expression profile datasets ( Figure 1E). Among these seven DEMs, miR-185-5p, named miR-185 previously (Supplementary Figure S1E), was the top overlapping DEMs and consistently downregulated in two datasets ( Figure 1F). Expression values of each sample in two datasets were listed in Supplementary Figure S1B,D. To confirm whether miR-185-5p changed in response to TLR4 signaling in vitro, we only used LPS, instead of LPS&INF-γ to specifically trigger TLR4 signaling in RAW264.7 mouse macrophage cell line. As the result, miR-185-5p was significantly downregulated by LPS ( Figure 1G).

miR-185-5p Regulates the Activation of MAPK Pathways
NF-κB and MAPK pathways play important role on LPS-activated inflammation in macrophages, we next examined the relation between miR-185-5p and the phosphorylation of key signaling proteins in NF-κB and MAPK pathways. Interestingly, miR-185-5p overexpression reduced the aberrant activation of phosphorylation in JNK, ERK1/2, and p38 ( Figure 4A,B) compared to miR NC. In parallel, knocking down miR-185-5p led to

miR-185-5p Regulates the Activation of MAPK Pathways
NF-κB and MAPK pathways play important role on LPS-activated inflammation in macrophages, we next examined the relation between miR-185-5p and the phosphorylation of key signaling proteins in NF-κB and MAPK pathways. Interestingly, miR-185-5p overexpression reduced the aberrant activation of phosphorylation in JNK, ERK1/2, and p38 ( Figure 4A,B) compared to miR NC. In parallel, knocking down miR-185-5p led to increased activation of JNK, ERK1/2, and p38 signaling pathways after LPS treatment ( Figure 4C,D). The LPS-induced phosphorylation of NF-κB p65 was not markedly changed by mediating miR-185-5p. These data suggest that miR-185-5p acts as a negative regulator in MAPK signaling pathway in LPS-activated macrophages.
Genes 2022, 13, x FOR PEER REVIEW 10 of increased activation of JNK, ERK1/2, and p38 signaling pathways after LPS treatment (F ure 4C,D). The LPS-induced phosphorylation of NF-κB p65 was not markedly changed mediating miR-185-5p. These data suggest that miR-185-5p acts as a negative regulator MAPK signaling pathway in LPS-activated macrophages.

CDC42 Is a Direct Target of miR-185-5p
Interaction with proteins is a major mechanism by which miRNAs regulate gene expression [31]. Therefore, to gain insights into the mechanism of miR-185-5p-regulated macrophage inflammation, downstream target genes of miR-185-5p were predicted by using ENCORI, miRWalk, and miRDB. We observed 16 overlapping genes ( Figure 5A). Among these, we focused on those known to be involved in MAPK pathways, and finally selected Cdc42 as a promising downstream candidate. To confirm the correlation between miR-185-5p and Cdc42, we transfected miR-185-5p mimics or miR-185-5p inhibitors and their normal controls in RAW264.7 macrophages. Although miR-185-5p failed to alter mRNA expression of Cdc42 ( Figure 5B), the protein level of CDC42 was negatively correlated with miR-185-5p expression ( Figure 5C-F).
The predicted miR-185-5p target sites at CDC42 3 UTR, shown in Figure 5G and Supplementary Figure S1F, were highly conserved across different species. We conducted dual luciferase report assays to verify the direct binding sites of CDC42 3 UTR and miR-185-5p. In Figure 5H, miR-185-5p mimics greatly reduced wild type CDC42 3 UTR luciferase activity, not mutants. These results indicate miR-185-5p can directly interact with CDC42 at protein level.

CASIN Modulates miR-185-5p-Mediated Pro-Inflammatory Phenotype Partially through Targeting MAPK JNK Pathways
Given the negative correlation between miR-185-5p and CDC42 described above, we hypothesized that miR-185-5p plays an anti-inflammatory role in macrophage by targeting CDC42. To test this, we used a selective small-molecule inhibitor of CDC42 named CASIN [32], to assess the effect of CDC42 inhibition on macrophage inflammation in RAW264.7 cells. CASIN rescued the mRNA level of Il1b and Il6 ( Figure 6A) in LPSactivated macrophages with miR-185-5p inhibitors transfection. Consistently, miR-185-5p inhibitors-mediated cytokine secretion of IL-1β and IL6 was also reversed by CASIN treatment ( Figure 6B). Furthermore, CASIN also reduced phagocytosis which was previously increased in LPS-induced macrophages with miR-185-5p suppression ( Figure 6C,D). Thus, miR-185-5p inhibitors promoted pro-inflammatory responses, and notably this effect was markedly reversed by inhibiting CDC42.

CASIN Modulates miR-185-5p-Mediated Pro-Inflammatory Phenotype Partially through Targeting MAPK JNK Pathways
Given the negative correlation between miR-185-5p and CDC42 described above, we hypothesized that miR-185-5p plays an anti-inflammatory role in macrophage by targeting CDC42. To test this, we used a selective small-molecule inhibitor of CDC42 named CASIN [32], to assess the effect of CDC42 inhibition on macrophage inflammation in RAW264.7 cells. CASIN rescued the mRNA level of Il1b and Il6 ( Figure 6A) in LPS-activated macrophages with miR-185-5p inhibitors transfection. Consistently, miR-185-5p inhibitors-mediated cytokine secretion of IL-1β and IL6 was also reversed by CASIN treatment ( Figure 6B). Furthermore, CASIN also reduced phagocytosis which was previously

Discussion
Macrophages are key players in atherosclerotic inflammation [31]. Emerging evidence implicates that miRNAs in LPS induced macrophage inflammation. However, the regulatory role of miR-185-5p in macrophage pro-inflammation has not been studied previously. In this study, we showed a novel anti-inflammatory role of miR-185-5p in vitro. Mechanically, miR-185-5p suppresses LPS-induced inflammatory response by partially targeting CDC42/JNK, consequently alleviating macrophage inflammation (Figure 7).

Discussion
Macrophages are key players in atherosclerotic inflammation [31]. Emerging evidence implicates that miRNAs in LPS induced macrophage inflammation. However, the regulatory role of miR-185-5p in macrophage pro-inflammation has not been studied previously. In this study, we showed a novel anti-inflammatory role of miR-185-5p in vitro. Mechanically, miR-185-5p suppresses LPS-induced inflammatory response by partially targeting CDC42/JNK, consequently alleviating macrophage inflammation (Figure 7).
We select miRNA profiling in Dicer knockout ApoE−/− mice, because Dicer is one of the key enzymes during miRNA biogenesis [33]. Macrophage-specific knockout of Dicer exhibits a pro-atherosclerotic phenotype with larger necrotic core in ApoE−/− mice. Pro-inflammatory gene expression including IL-1β and CCL2 is enhanced in lesional macrophages [26]. We observed that in absence of Dicer there was a huge decrease of miRNA expression in GSE87718, which is consistent with these findings in vitro Dicer knockout cells [34]. Young-Kook Kim and colleagues identify Dicer ablation can cause a depletion of 96% detected miRNA species [34]. miR-185-5p is significantly downregulated in the miRNA expression profiling of BMDMs in Dicer knockout compared to wildtype ApoE−/− mice.
Our results using mouse macrophages cell line confirmed an anti-inflammatory role for miR-185-5p. miR-185-5p mimics transfection decreased LPS-stimulated Il1b and Il6 in RAW264.7 macrophages. Previous reports show similar evidence that in human umbilical vein endothelial cells (HUVECs) LPS effectively decreases the expression of miR-185-5p, and miR-185-5p overexpression exhibits anti-inflammatory effects via inhibiting Il6 and Ccl2 [35]. IL-1β is a powerful mediator of atherosclerosis development. Once activated, IL-1β induces the expression of another pro-inflammatory cytokine IL6 in macrophages, triggering the synthesis of C-reactive protein (CRP) [36]. Targeting IL-1β-IL6-CRP axis with Canakinumab significantly prevents atherosclerosis-related cardiovascular events in multi-center clinical trial [37]. Macrophage inflammatory response alleviated by miR-185-5p suppresses the key driver of atherosclerotic inflammation. Conflicting study reports a pro-atherosclerotic role of miR-185-5p in ApoE−/− mice by negatively modulating hepatic cholesterol metabolism [25]. However, evidence in Wang and Zhan's study demonstrates miR-185-5p is protective toward cholesterol homeostasis in hepatocytes, consequently decreasing lipid synthesis in liver of C57BL/6 mice fed with high fat diet. Additionally, they indicate miR-185-5p increases insulin sensitivity by upregulating the insulin receptor substrate-2, which in turn activates the PI3K/AKT signaling [23]. We select miRNA profiling in Dicer knockout ApoE-/-mice, because Dicer is one of the key enzymes during miRNA biogenesis [33]. Macrophage-specific knockout of Dicer exhibits a pro-atherosclerotic phenotype with larger necrotic core in ApoE-/-mice. Proinflammatory gene expression including IL-1β and CCL2 is enhanced in lesional macrophages [26]. We observed that in absence of Dicer there was a huge decrease of miRNA expression in GSE87718, which is consistent with these findings in vitro Dicer knockout cells [34]. Young-Kook Kim and colleagues identify Dicer ablation can cause a depletion of 96% detected miRNA species [34]. miR-185-5p is significantly downregulated in the miRNA expression profiling of BMDMs in Dicer knockout compared to wildtype ApoE-/-mice.
Our results using mouse macrophages cell line confirmed an anti-inflammatory role for miR-185-5p. miR-185-5p mimics transfection decreased LPS-stimulated Il1b and Il6 in RAW264.7 macrophages. Previous reports show similar evidence that in human umbilical vein endothelial cells (HUVECs) LPS effectively decreases the expression of miR-185-5p, and miR-185-5p overexpression exhibits anti-inflammatory effects via inhibiting Il6 and Ccl2 [35]. IL-1β is a powerful mediator of atherosclerosis development. Once activated, IL-1β induces the expression of another pro-inflammatory cytokine IL6 in macrophages, triggering the synthesis of C-reactive protein (CRP) [36]. Targeting IL-1β-IL6-CRP axis with Canakinumab significantly prevents atherosclerosis-related cardiovascular events in multi-center clinical trial [37]. Macrophage inflammatory response alleviated by miR-185-5p suppresses the key driver of atherosclerotic inflammation. Conflicting study reports a pro-atherosclerotic role of miR-185-5p in ApoE-/-mice by negatively modulating hepatic cholesterol metabolism [25]. However, evidence in Wang and Zhan's study demonstrates miR-185-5p is protective toward cholesterol homeostasis in hepatocytes, consequently Mechanically, miRNAs have been identified to interact with 3 UTR of mRNAs thereby negatively regulating gene expression [33]. For example, miR-155 interacts with transcription factor Ets2 to modulate pro-inflammatory response in LPS-induced RAW264.7 [38]. We found miR-185-5p interacts with CDC42, which was consistent with previous findings [37]. CDC42 is critical for fetal survival that Cdc42-knockout mice are embryonic lethal [15,39]. CDC42 regulates diverse signaling pathways and cellular functions. The pro-migratory effect of CDC42 on endothelial cell and vascular smooth muscle cell has been demonstrated widely in atherogenesis [18,19]. Our novel results indicated that inhibition of CDC42 by sponging miR-185-5p effectively suppressed cytokine secretion and phagocytosis, which supported a pro-inflammatory role on CDC42. miR-185-5p might regulate CDC42 at posttranslational level. Post-translational modifications are important for regulating protein function, localization, and stability. More investigations are needed to uncover the underlying mechanism. CDC42 is involved in signaling to the JNK modules [9,17]. In our study, ablation of CDC42 with CASIN decreased the secretion of IL-1β and IL6 and suppressed the phosphorylation of JNK, even though phosphorylated ERK1/2 and phosphorylated p38 were augmented in miR-185-5p knocking down macrophages. Earlier findings show that silencing CDC42 significantly enhances phosphorylation of p38 and ERK1/2 in human skin fibroblasts [40]. The effect of CDC42 on macrophage inflammation is rather more complicated than we have expected. Therefore, the regulatory effect of miR-185-5p/CDC42 on inflammation resolution is partially dependent on JNK pathways, not ERK1/2 and p38 pathways.
TLR4 activation enhances phagocytic receptors [10]. We for the first time shows a negative role of miR-185-5p/CDC42 on macrophage phagocytosis. CDC42 is quite important for phagocytosis [41,42]. In a subline of RAW 264.7 cells, intact RAC1 and CDC42 function serves as essential effectors of phagocytosis [42]. However, phosphoinositide 3-kinase enables phagocytosis of large particles in macrophages through inactivating Rac/Cdc42 [43]. In addition, the effect of phagocytosis is quite complicated in atherosclerosis. Enhanced phagocytosis improves lipid uptake and foam cell formation, enables lesion formation, which is protective in the early stage of atherosclerosis [31]. In advanced atherosclerosis, incomplete clearance and defective phagocytosis give rise to necrotic cores, which might contribute to plaque instability and plaque rupture [44]. Thus, further in vivo studies are needed to determine whether miR-185-5p plays diverse role in different stages of atherosclerosis.
Although we have proved that miR-185-5p can directly target CDC42, miRNA regulates cell functions through multitargets. There are also other miR-185-5p-regulated targets that existed in macrophage inflammatory activation. We found p21-activated protein kinases PAK4 may be another potential target for miR-185-5p with high prediction score in the predicting list of TargetScan. PAK4 has been proved to be a switch between caspase-8modulated apoptosis and NF-κB in TNF-α-induced hepatocarcinoma cells [45]. We could not rule out other potential mechanisms involved in miR-185-5p-mediated macrophage inflammation. On the other hand, our study has some limitations. We demonstrated the effect of miR-185-5p in macrophage phagocytosis in vitro. Phagocytosis is an important function in macrophage and plays diverse roles in different stages of atherosclerosis [31]. In vivo study is urgently needed to illustrate whether miR-185-5p has an effect on macrophages infiltration and lesional inflammation in atherosclerosis. In addition, whether the regulatory effects of CDC42 on ERK1/2 and p38 are involved in atherosclerotic inflammation is not clear. In previous findings it was shown that, CDC42-ERK1/2 interaction is related to CCL2 secretion, which is a strong chemokine in macrophage recruitment [40]. The role of miR-185-5p on CCL2 expression needs further investigation.

Conclusions
In this study we showed that miR-185-5p exhibited anti-inflammatory functions at least in part through targeting CDC42/JNK pathways in LPS-induced macrophages. These findings yield insights into the understanding of miR-185-5p-regulated network in macrophages inflammation, which is beneficial for exploring the mechanism of macrophage inflammation in atherosclerosis.