MiR-19a-3p Suppresses M1 Macrophage Polarization by Inhibiting STAT1/IRF1 Pathway

Macrophages, an important type of immune cells, are generally polarized to classically activated macrophage (M1) or alternatively activated macrophage (M2) to respond to environmental stimuli. Signal transducer and activator of transcription 1 (STAT1), a very important transcription factor, can promote M1 macrophage polarization. However, the mechanisms of regulating STAT1 in macrophage polarization remain unclear. In the present study, STAT1 was markedly elevated, however, miR-19a-3p was down-regulated in interferon (IFN)-γ and lipopolysaccharide (LPS) treated RAW264.7 cells, and dual-luciferase reporter assay identified that miR-19a-3p directly targeted STAT1 by binding to its 3′UTR. Up-regulated miR-19a-3p inhibited M1 polarization by targeting STAT1/interferon regulatory factor 1 (IRF1) and vice versa in vitro. Consistently, overexpression of miR-19a-3p in LPS treated mice by systemically administering agomiR-19a-3p effectively reduced the inflammation in mouse lung tissues, and inhibited M1 macrophage polarization via suppressing STAT1/IRF1 pathway. In summary, our study confirmed that miR-19a-3p, as a direct regulator of STAT1, inhibited M1 macrophages polarization. The miR-19a-3p/STAT1/IRF1 pathway can potentially be used to design novel immunotherapy for modulating macrophage polarization.


INTRODUCTION
Macrophages, an essential component of innate immunity, have high plasticity and can display divergent phenotypes and functions (Lawrence and Natoli, 2011). In respond to different environmental stimuli, macrophages can develop into classically activated macrophages (M1 type) and alternatively activated macrophages (M2 type) (Franklin and Li, 2016;Cassetta and Pollard, 2020;Larionova et al., 2020b). M1 macrophages play a prominent role in immune surveillance by secreting pro-inflammatory cytokines and chemokines, and high antigen presentation. While M2 macrophages have significant effects in immune regulation by secreting cytokines IL-10 and/or TGF-β which are related to anti-inflammatory effect (Lawrence and Natoli, 2011;Hamilton et al., 2014;Bi et al., 2016;Shapouri-Moghaddam et al., 2018). Many transcription factors play important roles in macrophage polarization, such as interferon regulatory factors (IRFs), nuclear transcription factor-κB (NF-κB), signal transducer and activator of transcriptions (STATs), and so on (Lawrence and Natoli, 2011;Glass and Natoli, 2016;Huang et al., 2018;Li et al., 2018;Ma et al., 2018;Larionova et al., 2020a). Among them, STAT1 exerts a crucial role in modulating the polarization of macrophages. STAT1, a major member of the STAT family, is activated by janus kinase (JAK) upon the stimulation of interferon (IFN)-γ (Osterlund et al., 2007). Activated STAT1 homodimers transfer into the nucleus and promote IFN-γ-response genes related to anti-virus, antigen present, and microbicidal function (Hu and Ivashkiv, 2009;Wynn et al., 2013). STAT1 is involved in cell differentiation, function and apoptosis processes, as well as tumor, chronic inflammation and autoimmune diseases (Li et al., 2015a;He et al., 2016;Dufour et al., 2018;Wang and Zhu, 2019). More recently, several researches uncovered the effects of STAT1 in macrophage polarization, and underline the significance of the STAT1-mediated macrophages' activation (Kernbauer et al., 2012;Juhas et al., 2015). In human primary macrophage, RAW264.7 and THP-1 cells PARP14 silencing promoted IFN-γ-induced STAT1 activation and promoted pro-inflammatory macrophage polarization (Iwata et al., 2016). Ning Ding et al. pointed out that STAT1 played a significant role in M1 polarization, whereas physalin D suppressed the STAT1 activation to inhibit M1 polarization (Ding et al., 2019). These studies discover that STAT1 plays key roles in M1 polarization, while the up-stream factors that regulate STAT1 expression are poorly understood.
MiR-19a-3p, a member of the miR-17-92 miR cluster, is located on human chromosomes 13 and mouse chromosomes 14, respectively. A large number of studies have shown that miR-19a-3p is involved in the occurrence and development of a variety of cancers, including breast cancer, lung cancer, gastric cancer, hepatocellular carcinoma and so on (Wang et al., 2013a;Li et al., 2014;Lu et al., 2014;Li et al., 2015b;Tan et al., 2015;Ma et al., 2016;Feng et al., 2018;, and high levels of serum miR-19a-3p can be used as an independent prognostic indicator in patients with non-small cell lung cancer and esophageal squamous cell carcinoma (Lin et al., 2013;Xu et al., 2014). Meanwhile, researches have reported miR-19a-3p is related to proliferation, apoptosis, metastasis, and chemoresistance process of malignant tumors (Yang et al., 2014;Ma et al., 2016;Jiang et al., 2018;. These studies indicate that miR-19a-3p is involved in a variety of physiological and pathological processes, but the role of miR-19a-3p in macrophage polarization has not been fully elucidated. Recently, a growing number of researches have indicated that STAT1 can be controlled by some miRNAs. For example, upregulated miR-146a can reduce NK cell-mediated cytotoxicity and the expression of TNF-α and IFN-γ by targeting STAT1 (Xu et al., 2017), and depresses T-cell immune function in chronic hepatitis B (CHB) patients by inhibiting STAT1 (Wang et al., 2013b). Moreover, enhancing the expression of miR-30a in subcutaneous fat pads of diabetic mice can increase insulin sensitivity, energy consumption, and reduce liver ectopic fat deposition and inflammation of white adipose tissue by inhibiting STAT1 signaling pathway (Koh et al., 2018). However, the mechanisms of miRNAs in macrophage polarization through targeting STAT1 are still unclear.
In this study, microarray analysis showed that miR-19a-3p was reduced, while STAT1 was increased significantly in M1 macrophages derived from RAW264.7 cells, and miR-19a-3p was negatively correlated with STAT1. Overexpression of miR-19a-3p suppressed M1 polarization whereas inhibition of miR-19a-3p showed the opposite result in vitro. Moreover, overexpressed miR-19a-3p reduce lung inflammation by depressing M1 macrophages in mice treated with LPS. Furthermore, we found that miR-19a-3p suppressed M1 polarization via inhibiting STAT1/IRF1 pathway by targeting STAT1. Our study confirmed that miR-19a-3p, as a direct regulator of STAT1, affected M1 macrophage polarization for the first time. and unstimulated RAW264.7 (n 3) were used for microarray analysis. The sample treatment, data acquirement and analyzation were performed as previously described . The microarray data have been deposited in Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih. gov/geo) (GSE143845).

RNA Isolation and Quantitative
Real-Time PCR RNA isolation from cells and mouse tissues and qRT-PCR were similar as our previous publication using commercial kits . Briefly, Total RNA were purified from cells and tissues by TRIzol Reagent and converted to miRNA and mRNA cDNA by the miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and the PrimeScript RT reagent Kit (Toyobo, Osaka, Japan) respectively. To amplify miRNA and mRNA, SYBR Green was used and qRT-PCR was performed on Applied Biosystems 7500 instrument. The amplification reaction for each sample was performed in triplicate. Relative quantification was calculated by the comparative 2 −ΔΔCt method. The primer sequences are displayed in Table 2.

Luciferase Reporter Assay
Dual luciferase reporter assay was carried out as previous described . In brief, the wild-type (WT) or mutant (MUT) STAT1 3′UTR was cloned into pGL3-3M-Luc vector (Promega, Madison, WI). 293T cells were co-transfected with WT or MUT luciferase reporter plasmid and 100 nM mimics or NC of miR-19a-3p for 24 h. Luciferase activity in the cells was analyzed using the Dual-Luciferase ® Reporter Assay System (Promega) on GloMax 20/20 Luminometer (Promega). Relative luciferase activity was standardized to the renilla luciferase luminescence.

Animal Experiments
Eight-week-old C57BL/6 mice were obtained from Beijing Biotechnology Co., Ltd., housed in the specific pathogen-free facility. The animal study was approved by the Animal Ethics Committee of Shandong First Medical University (No. SDFMU2018-A04) and performed in accordance with the National Academies Guiding Principles for the Care and Use of Laboratory Animals, 8th edition. Mouse (n 10 per group) was injected with 100 μl saline or saline containing 10 nM miR-19a-3p agomiR (Ribobio, Guangzhou, China) or 10 nM agomiR NC (Ribobio) by tail vein for 1 h, and then treated with LPS (20 mg/kg) by intraperitoneal injection, as described in a previous study . After 24 h of LPS treatment, blood sampling in mice eyes were collected. After centrifugation at 3,000 rpm for 10 min, mouse plasma was collected and stored at -80°C for future experiments. Peritoneal macrophages were immediately collected as previously described (Ray and Dittel, 2010). In brief, mice were intraperitoneally injected with 5 ml ice-cold 1 × PBS containing 3% fetal bovine serum (FBS) followed by massage for 5 min. Peritoneal fluid was collected into 15 ml sterile centrifuge tube followed by centrifuging at 1,500 rpm/min for 8 min. Peritoneal macrophage were cultured in DMEM containing 10% FBS at 37°C with 5% CO 2 . Then, the supernatant was removed at 4 h, and the cells were washed with DMEM slowly to remove non-adherent cells. The purity of peritoneal macrophages was detected by flow cytometry using APC-conjugated antimouse F4/80 (17-4801-82, eBioscience) antibody. After collecting peritoneal macrophages, the lungs of mice were immediately collected.

Hematoxylin and Eosin Staining
Lung tissues were fixed in 4% paraformaldehyde and made into 5 μm paraffin slices. Sections were then baked at 60°C for 1 h and dewaxed by xylene. After hydration, tissue sections were stained with H&E (Solarbio) following standard procedures, then dehydrated with alcohol gradients followed by clearing using xylene. All histological images were obtained by an optical microscope (E100; Nikon, Japan).

Statistical Analysis
All experiments were performed at least three independent times unless otherwise stated. Values are listed as the mean ± SEM. The 2-tailed Student t test was applied to calculate the statistical significance between two different groups. One-way ANOVA followed by Bonferroni test were used for multiple comparisons. Correlations were analyzed by Pearson correlation. Data were plotted using GraphPad Prism 6.0 software (GraphPad Software, Inc., CA, United States). Statistical analyses were carried out by SPSS software (version 16.0). p < 0.05 was considered significant.

STAT1 is a Direct Target Gene of miR-19a-3p
To study the direct binding of miR-19a-3p with STAT1, plasmid with WT or MUT 3′UTR of STAT1 were constructed ( Figure 6A), which was co-transfected with miR-19a-3p mimics or NC for dual-luciferase reporter assay. It was found that miR-19a-3p mimics significantly decreased luciferase reporter activity of WT 3′UTR plasmid of STAT1 ( Figure 6B), but didn't influence that of MUT 3′UTR plasmid ( Figure 6C). These data above indicated that STAT1 was a direct target gene of miR-19a-3p.
Frontiers in Pharmacology | www.frontiersin.org May 2021 | Volume 12 | Article 614044 6 J mice were treated with 20 mg/kg LPS or saline by intraperitoneal injection, after 24 h, we found that challenged with LPS increased the infiltration of inflammatory cells obviously in lung tissues compared with control mice ( Figure 7A). MiR-19a-3p was down-regulated in the peritoneal macrophages of mice that challenged with LPS ( Figure 7B). Meanwhile, TNF-α, CXCL9, CXCL10 were increased in the peritoneal macrophages after treated with LPS   Figures 7C,D). In addition, LPS treated up-regulated the expression of STAT1 and IRF1 ( Figures 7E,F). These results suggested that M1 macrophage increased obviously in the LPS challenged sepsis model, and miR-19a-3p regulated STAT1/IRF1 signaling pathway is involved in this process.
To confirm that the STAT1/IRF1 signal pathway regulated by miR-19a-3p plays an important role in M1 macrophage polarization in LPS challenged sepsis model. Male C57BL/6 J mice were administered with 10 nM agomiR-19a-3p or agomiR NC by tail vein injection, and then treated with 20 mg/kg LPS by intraperitoneal injection. The pre-treatment of agomiR-19a-3p significantly reduced the infiltration of inflammatory cells in the lung tissues compared with that in agomiR NC groups ( Figure 8A). MiR-19a-3p was increased in the peritoneal macrophages of the agomiR-19a-3p group ( Figure 8B), in which TNF-α, CXCL9, CXCL10 decreased obviously compared with that in agomiR NC group (Figures 8C,D). Meanwhile, STAT1 and IRF1 were obviously reduced in agomiR-19a-3p group (Figures 8E,F). These data proved that miR-19a-3p could inhibit M1 macrophage polarization by STAT1/IRF1 pathway in vivo.
FIGURE 6 | STAT1 is a direct target of miR-19a-3p. (A) Schematic representation of predicted binding sequence between the 3′UTR of STAT1 and miR-19a-3p, wild-type (WT) and mutant (MUT) STAT1 plasmid construction. (B,C) Transcriptional activity of STAT1 was determined by the dual-luciferase reporter assay in 293T cells (data were pooled from three independent experiments). Each sample had three replicates. Statistical significance was calculated using unpaired Student's t-test. *p < 0.05, ns means no statistical difference.
Frontiers in Pharmacology | www.frontiersin.org May 2021 | Volume 12 | Article 614044 9 FIGURE 7 | M1 macrophage increased obviously in the LPS challenged sepsis model. Male 5C7BL/6 J mice were challenged with 20 mg/kg LPS or saline by intraperitoneal injection for 24 h. (A) H&E staining of lung tissues treated with LPS and control mice (a representative experiment, from three independent experiments) (Scale bar, 50 μm, 200×). (B,C) miR-19a-3p, TNF-α, CXCL9, and CXCL10 in the peritoneal macrophages of mice treated with LPS and control mice were determined using qRT-PCR (data were pooled from three independent experiments with six mice per group). (D) TNF-α, CXCL9, and CXCL10 in serum of mice treated with LPS and control mice were measured by ELISA (data were pooled from three independent experiments with six mice per group). (E) STAT1 and IRF1 were detected in the peritoneal macrophages of mice treated with LPS and control mice (data were pooled from three independent experiments with six mice per group). (F) IRF1 and STAT1 were measured by western blot in the peritoneal macrophages of mice treated with LPS and control mice (a representative blot, from three independent experiment with three mice per group). Statistical significance was calculated using unpaired Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
FIGURE 8 | Overexpressed miR-19a-3p reduced the inflammation of lung tissues and inhibited M1 polarization in LPS-treated mice. Male 5C7BL/6 J mice were treated with agomiR-19a-3p or agomiR NC for 1 h, followed by the 20 mg/kg LPS challenge by intraperitoneal injection. (A) H&E staining of lung tissues of mice treated with agomiR-19a-3p or agomiR NC followed by challenging LPS (a representative experiment, from three independent experiments). (Scale bar,50 μm,200×). (B,C) miR-19a-3p, TNF-α, CXCL9, and CXCL10 in the peritoneal macrophages of mice using qRT-PCR (data were pooled from three independent experiments with ten mice per group). (D) TNF-α, CXCL9, and CXCL10 in serum of mice were measured by ELISA (data were pooled from three independent experiments with ten mice per group). (E) STAT1 and IRF1 were detected in the peritoneal macrophages of mice (data were pooled from three independent experiments with 10 mice per group). (F) IRF1 and STAT1 were measured by western blot in the peritoneal macrophages of mice (a representative blot, from three independent experiment with six mice per group). Statistical significance was calculated using unpaired Student's t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001. Increasing evidence has shown that the polarization of macrophages is regulated by a few transcription factors (Juhas et al., 2015;Glass and Natoli, 2016;Huang et al., 2018;Li et al., 2018;Larionova et al., 2020a). Among these transcription factors, STAT1 plays key roles in modulating macrophage polarization. STAT1 belongs to the STATs family and is a prototypical member of the JAK/STAT pathway (Aaronson and Horvath, 2002). STAT1 signaling pathway is involved in the cross-talk between innate and adaptive immunity (Hu and Ivashkiv, 2009;Sikorski et al., 2012;Chmielewski et al., 2014). It was reported that ferric ammonium citrate treatment could inhibit RAW264.7-derived M1 macrophage by suppressing STAT1 (Gan et al., 2017). Chrissy M. Leopold Wager et al. indicated that STAT1 played key roles in M1 macrophage polarization and NO production during Cryptococcus neoformans infection (Leopold Wager et al., 2015). In our study, it was found that both total and phosphorylated STAT1 were obviously increased in IFN-γ and LPS induced M1 macrophages that were consistent with previous results (Iwata et al., 2016). However, the upstream mechanisms of regulating STAT1 in macrophage polarization remain unclear.
Firstly, it should be noticed that the expression profiles of miRNAs and genes expressed differently in different induction conditions. Secondly, the underlying mechanisms of macrophage polarization regulated by miRNAs are diverse, which might involve various target genes and cross-talk among miRNAs. Therefore, in different induced conditions, miR-19a-3p may regulate different genes and then produce different regulatory effects. In summary, the present study reveals for the first time that overexpression of miR-19a-3p can suppress M1 macrophage polarization both in vitro and in vivo. MiR-19a-3p downregulates STAT1 expression by targeting STAT1 3′UTR, thus inhibits IRF1 expression and M1 macrophage phenotype polarization ( Figure 9). The miR-19a-3p/STAT1/IRF1 axis may be a potential target in macrophage polarization, and highlights a better comprehension for potential mechanism of pathogenesis and progression of diseases such as chronic inflammatory and autoimmune diseases. Our findings may provide promising biomarkers for prognosis of cancer patients, as well as therapeutic targets to design novel immunotherapy for the treatment of cancers including lung cancer. Meanwhile, it should be noticed that since the regulation of macrophage polarization and function is complicated, the role of miR-19a-3p on macrophage still needs further in-depth research.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The datasets presented in this study can be found in NCBI using the accession GSE143845.

ETHICS STATEMENT
The animal study was reviewed and approved by School of Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences.