Immunopharmacology and Immunotoxicology

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Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro Yang Yang, Ben-Quan Wu, Yan-Hong Wang, Yun-Feng Shi, Jin-Mei Luo, JunHui Ba, Hui Liu & Tian-Tuo Zhang To cite this article: Yang Yang, Ben-Quan Wu, Yan-Hong Wang, Yun-Feng Shi, Jin-Mei Luo, Jun-Hui Ba, Hui Liu & Tian-Tuo Zhang (2016): Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro , Immunopharmacology and Immunotoxicology, DOI: 10.1080/08923973.2016.1248845 To link to this article: http://dx.doi.org/10.1080/08923973.2016.1248845

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Date: 19 October 2016, At: 22:49

Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro Yang Yang1 , Luo1, 1

Ben-Quan Wu1*,

Jun-Hui Ba1,

Yan-Hong Wang1,

Yun-Feng Shi1, Jin-Mei

Hui Liu1, Tian-Tuo Zhang1

Department of Internal Medicine, Medical Intensive Care Unit and Division of

Respiratory Diseases, Institute of Respiratory Disease and the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, People’s Republic of China

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*Corresponce author: Ben-Quan Wu address:Department of Internal Medicine,

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Medical Intensive Care Unit and Division of Respiratory Diseases, Institute of Respiratory Disease and the Third Affiliated Hospital, Sun Yat-Sen University,

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Guangzhou 510630, People’s Republic of China e-mail: [email protected]

Abstract Macrophages play an important role in inflammatory responses; however, miRNA-mediated repolarization of macrophages is essential for fulfilling this function. To clarify a series of changes at the RNA level in alveolar macrophages under normal and inflammatory conditions, bronchial alveolar lavage liquid (BALF) was collected from healthy volunteers or patients with pneumonia. This approach, which differs from that used in previously, provides more accurate information about

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the states of macrophages in different lung microenvironments. In this study, the

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density plots of macrophage subtypes (M1 and M2) in the BALF of healthy

volunteers differed from that of the patients with pneumonia. The M2 subtype

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dominated in healthy volunteers and was rapidly repolarized to M1 in response to

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miRNA-mediated gene regulation. Differential miRNA expression in the two macrophage subtypes revealed lower expression of miR-155 and MIR-146a in patients with pneumonia compared with healthy volunteers; this may be related to inflammation and the use of anti-inflammatory drugs. We also found increased TNF-α

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and IL-6 expression at the RNA level, while macrophage galactose-type C-type lectin

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1 (MGL-1) expression decreased with downregulation of miR-155 and miR-146a expression. These results indicate that the gene regulation mediated by miR-155 and miR-146a contributes to human alveolar macrophage phenotype repolarization, thus leading to an early switch from pro-inflammatory to anti-inflammatory cytokine production. Keywords:Macrophages, MiRNA, Repolarization, Pneumonia

Introduction Macrophages constitute the first line of host defense during infection and, therefore, play a crucial role in the early recognition and subsequent triggering of pro-inflammatory responses to invading pathogens. Certain components of pathogens and cytokines initiate the process of macrophage polarization. Different micro-environmental factors stimulate macrophages to polarize into distinct subsets, including the classic activation (M1) and selective activation (M2) subsets, which

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express different surface receptors, cytokines, chemokines and intracellular enzymes

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[1]. The polarization of macrophages is plastic, rapid and fully reversible.

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MiRNAs are small (22-bases) non-coding RNAs encoded by a member of a family of endogenous RNAs, which regulate target mRNAs after transcription. In this way,

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miRNAs mediate transcriptional regulation of the expression of a variety of genes relating to physiological functions of many animals and plants. Currently, the function of miRNAs in pathogenic infections has become a focus of research. Activated

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macrophages control pathogen-induced inflammation by producing specific miRNAs[2]. Many studies show that miRNAs play an important role in inflammatory

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cytokine-induced inflammation in response to pathogens; for example, miR-146a and miR-155 are upregulated in LPS-activated macrophages[3, 4]. However, Moschos et al. reported significant upregulation of miRNA-21, -25, -27b, -100, 140, -142-3p, -181c, 187, -194, -214, -224 and -223, but not miRNA146 or miRNA-155, in mouse macrophages activated by LPS over a different time-frame. This suggested that miRNA expression is related to infectious duration[5]. In addition, miRNA expression

and the mechanism of regulation is also cell type-dependent [6]. The dominance of the M1 or M2 subset and the types of cytokines secreted are influenced by numerous factors, including the type of macrophages and conditions such as viral infections and accompanying diseases (e.g. asthma) [7]. In multiple myeloma, conditions in which M1 is repolarized to M2 are likely to affect the damage and repair of bone, as well as to inhibit tumor growth [8, 9]. Previous studies show that the polarization and functional status of macrophages is closely related to miRNA

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expression, although the underlying mechanism is not clear [10]. In addition, the

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expression of miRNAs may vary depending on species differences and the in vitro or in vivo conditions of the investigation [5]. For example, the expression patterns of

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arginase (Arg1), nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in

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human and murine macrophages are distinct; human macrophages do not express NO and iNOS, and Arg1 is expressed only in human neutrophils, although all three are expressed in murine macrophages. The miRNA expression patterns of human alveolar macrophages have not yet been elucidated; therefore, in this study, we investigated the

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distribution and function of human alveolar macrophages from healthy volunteers and

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patients with pneumonia. We also investigated the regulatory effects of miRNAs on the secretion of inflammatory cytokines by macrophages in vitro.

Materials and methods Healthy volunteers and patients with pneumonia Healthy volunteers were recruited according to the following inclusion criteria: aged 18– 65 y, non-smoker, no respiratory diseases, no hematopathy and immune systems or other underlying diseases. No infectious diseases or use of antibiotics, immunosuppressants or steroid hormones within 1 month before surgery. Routine blood tests, liver and kidney function, blood coagulation tests, electrocardiography

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(ECG), chest radiography and bacteriological examination from throat swabs were

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normal within 1 month before surgery.

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Patients with pneumonia were recruited according to the following inclusion criteria: aged 18– 65 y, non-smoker, no respiratory diseases, no hematopathy and

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immune systems or other underlying diseases. No use of immunosuppressants or steroid hormones within 1 month before surgery. Blood coagulation tests and ECG examination were normal. Bacterial pneumonia confirmed by bacteriological tests of

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throat swabs or bronchial alveolar lavage liquid (BALF) and radiological examination.

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All experiments using human samples were approved by the Medical Ethics Committee of Sun Yat-sen University. Informed consent was obtained from each person participating in this study.

Collection of BALF by bronchoscopy Bronchoscopy of the lingular bronchus of the left lung and the middle lobe of the

right lung (in areas of inflammation) was performed using three successive aliquots (30 ml each) of sterile saline. The BALF aspirated from lungs was collected into sterile silicified cryogenic containers and transported (0°C) to the laboratory for cell separation.

Isolation of macrophages The BALF was filtered through four layers of sterile gauze in lamina flow cabinet

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and then centrifuged at 1,200 r/min for 10 min (4°C). The pelleted cells were

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resuspended, washed twice with 5 ml of PBS (containing 100 U/ml penicillin and 100 μg/ml streptomycin; GIBCO, USA) and centrifuged 1,200 r/min for 10 min (4°C).

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The cells were resuspended in complete RPMI 1640 (containing 10% fetal bovine

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serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin; GIBCO).

Culture of macrophages

After separation, cells were added to 24-well plates (1 × 106 cells/well) and

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incubated at 37°C under 5% CO2. After 4 to 6 h, the non-adherent cells were washed

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off with pre-warmed (37°C) incomplete RPMI 1640 (without FBS; GIBCO). The adherent cells were then removed by gentle pipetting and complete RPMI 1640 medium was added to continue the culture of alveolar macrophages for subsequent experiments.

Repolarization of human macrophages Macrophages were isolated and cultured as described previously prior to classical stimulation (1) by 1 μg/ml of LPS (Sigma-Aldrich, St. Louis, USA) and 10 ng/ml of IFNγ (Peprotech, Rocky Hill, USA) or alternative stimulation (2) by 10 ng/ml of IL4 and 10 ng/ml of IL13 (both Peprotech or no stimulation (3) (PBS control). Cells were then incubated at 37°C for 24 h. Total RNA was then extracted for analysis.

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RNA isolation and cDNA synthesis

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Total RNA was isolated using TRIzol reagent (Life Technologies, Inc.,

Gaithersburg, MD, USA) according to the manufacturer’s instructions and

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re-dissolved in 30–50 µl DEPC-treated water. Samples were then stored at -80°C for

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subsequent experiments. First strand cDNA was generated by reverse transcription with M-MLV reverse transcriptase (Life Technologies, Inc.).

qRT-PCR

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SYBR® Select Master Mix and TaqMan® Universal Master Mix II (with uracil

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N-glycosylase) were used for qRT-PCR. All TaqMan PCR analyzed by using the following conditions: 10 minutes at 95 ℃ after, at 95 ℃ for 15 seconds, 60 ℃ for 1 min, this process is carried out in 40 cycles.Human β-actin or U6 small nuclear RNA (U6 snRNA) was used as an endogenous control for normalization. All specific SYBR Green primers were verified before use and the sequences are as follows: MGL-1 (forward: 5'-AGGGTTTCAAGCAGGAACG-3', reverse:

5'-AGGTGTGCCTTCTGCGTAGT -3'), IL-6 (forward: 5'-ACTTGCCTGGTGAAAATCAT-3', reverse: 5'-CAGGAACTGGATCAGGACTT-3'), TNF-α (forward: 5'-ACTTGCCTGGTGAAAATCAT-3', reverse: 5'-CAGGAACTGGATCAGGACTT-3'), and β-actin (forward: 5'-AGCGGGAAATCGTGCGTGAC-3', reverse: 5'-TCCASTGCCCAGGAAGGAAGG-3'). The average relative difference in

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expression level of genes were calculated using the 2-ΔΔCt approach.

Transfection

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Human alveolar macrophages were grown in 24-well plates in complete medium

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without antibiotics for 24 h before transfection. At approximately 40% to 60% confluence the cells were transfected with siRNA (GenePharma, Shanghai, China) targeting miRNA155 and miRNA146a using Lipofectamine RNAi MAX (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. 24h

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later, we used cell scrapers to scrape the cells from 24-well plates and collected the

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cells for following experiments.

Flow cytometry

Cells were incubated for 30 min at room temperature with the antibodies for the detection of CD80 (FITC BD Pharmingen USA) CD163 (Alexa 647 BD Pharmingen USA) and an isotype-matched control (BD or eBioscience). The cells were then

washed three times with 10% FBS-containing PBS (centrifuged for 1,000 rev/min for 3 min) to block non-specific binding sites, and analyzed using Cell Quest software FACS Calibur.

Statistical analysis Statistical analysis was carried out by SPSS 20.0 software. All data were expressed as mean ± SEM. The differences between groups were analyzed by Student’s t-test or

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Mann-Whitney rank sum tests. P < 0.05 was considered to indicate statistical

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significance.

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Results

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The distribution of alveolar macrophage phenotypes in healthy volunteers and patients with pneumonia

M1 and M2 macrophages were analyzed by flow cytometric detection of the surface markers, CD80 and CD163, respectively[11-16]. Compared with healthy volunteers,

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alveolar macrophages from patients with pneumonia showed significantly lower

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CD163 expression, while there was almost no difference in the expression of CD80 between the two groups (FIG. 1 A,B). This suggested that the M2 subtype predominated in healthy lungs. The distribution of alveolar macrophage subtypes in patients with pneumonia was different from that in healthy volunteers (FIG. 1C). In the lungs of patients with pneumonia, the frequency of M2 macrophages decreased sharply, while that of the M1 macrophages appeared to increase.

Repolarization of human alveolar macrophages To verify repolarization of human alveolar macrophages in vitro, specific cytokines were added to the cell culture medium to stimulate the cells to repolarize. The morphology of the macrophages in culture changed during stimulation. The parapodium was extended in some of the M1 macrophages, but while almost no changes were observed in the M2 group (FIG. 2 A,B,C,D). Total RNA was isolated

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from alveolar macrophages after repolarization for 24 h to detect the expression of

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TNF-α, macrophage galactose-type C-type lectin 1 (MGL-1) ,IL-6, miR-155 and miR-146a. TNF-α, IL-6 and IL-12 reflect M1 polarization and MGL-1 reflects M2

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polarization [17-21]. After 24 h of stimulation with IFN-γ/LPS, expression of TNF-α

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and IL-6 increased (FIG. 3A,C), while MGL1 expression increased significantly after stimulation IL-4/IL-13 for the same period (FIG. 3B). These data showed that LPS/IFN-γ stimulated repolarization of the human alveolar macrophages from M2 to M1, while IL-4/IL-13 stimulated repolarization to M2. Expression of TNF-α and IL-6

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increased initially during the repolarization, although the expression of both cytokines

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then decreased after 24 h(FIG.3 A,C). After 48 h, IL-12 expression also increased in M1 macrophages (FIG.3 D)(P < 0.05). It showed that the alveolar macrophages derived from pneumonia patients could respond to either M1 or M2-polarising stimuli the same way as the ones from heathy donors (FIG. 3E). Investigation of the effects of the repolarization on miRNA expression in macrophages showed that both miR-155 and miR-146a increased after repolarization when M2 was repolarized to

M1 (FIG. 3F). Our data showed that miR-155 and miR-146a was both lower after repolarization at 48h than 24h, but the change of miR-146a expression had no statistical significance(FIG. 3F).

MiRNA expression in alveolar macrophages from healthy volunteers and patients with pneumonia The distribution of macrophage types was different in healthy volunteers compared

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with that in patients with pneumonia. Previous studies have shown that miRNA-155

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and miRNA-146a are closely related to macrophages[3, 4, 10, 22]; therefore, we hypothesized that there are differences in the expression of these miRNAs between

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healthy volunteers and patients with pneumonia. We found that the expression of

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miRNA-155 and miRNA-146a was reduced in patients with pneumonia compared with that in healthy volunteers(FIG. 4,*P < 0.05). This may be related to the impaired function of the immune system in the later stages of inflammation and pneumonia and the use of therapeutic drugs.

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The repolarization of human alveolar macrophages was induced in the presence of

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inhibitors of miR-155 or miR-146a Human alveolar macrophages were transfected with inhibitors of miR-146a or

miR-155 and the RNA levels of TNF-α, IL-6 and MGL-1 were then analyzed. The results showed that when miR-155 or miR-146a was inhibited, IL-6 and TNF-α increased while MGL-1 decreased in patients with pneumonia compared with healthy volunteers(FIG.5A,B,C, *P < 0.05). The results suggested that miR-146a and

miR-155 might be related to the anti-inflammatory activity of macrophages. Furthermore, miR-146a and miR-155 are implicated in mechanism of M1 and M2 repolarization.

Discussion Previous studies have demonstrated that M1-type macrophages can kill bacteria effectively, while the M2-type does not perform this function[1, 23, 24]. Our studies

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showed that the M2-type alveolar macrophages predominated in BALF in healthy

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volunteers, and the frequency of M2 macrophages decreased sharply, while that of the M1 macrophages appeared to increase in patients with pneumonia. We also

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found that human alveolar macrophages have the potential to repolarize from M1 to

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M2 following in vitro stimulation [25, 26]. Our analysis of miR-155 and miR-146a expression showed that macrophages were rapidly repolarized from one subset to another. Moreover, after inhibition of miR-155 or miR-146a, corresponding changes in TNF-α, IL-6, and MGL-1 expression were observed, suggesting that macrophages

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are repolarized by regulation of miR-155 or miR-146a, which influences the

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progression of pneumonia.

Previous studies have shown that the M1 and M2 polarization status in lungs is

related to some bacterial infections, cytokine expression and some diseases such as acute lung injury (ALI)[27, 28]. However, both M1 and M2 decreased in patients with pneumonia, indicating that certain stages of pneumonia or other immune cells lead to decreased macrophage frequencies. Our data also demonstrated the plasticity of

macrophages from healthy volunteers and patients with pneumonia, which were repolarized rapidly following in vitro stimulation (24 h). It was shown that the alveolar macrophages responded to either M1 or M2-polarising stimuli the same way as the ones from heathy donors. However, differences in the time required for this process between the M1 and M2 subtypes were observed. In the process of pneumonia, repolarization of alveolar macrophages may occur before other cells are recruited to the site of infection[29], although the changes in alveolar macrophage

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function at different stages of this process remain to be elucidated.

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Previous studies have indicated that miR146a and miR-155 in macrophages contribute to the regulation of LPS-stimulated inflammatory responses [3, 4, 30]. In

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accordance with this, we found that expression of miR-146a and miR-155 decreased

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in patients with pneumonia. Another recent study revealed upregulated miR-155 expression in patients with post-viral bacterial pneumonia [31], indicating that miR-155 expression is altered in different types of pneumonia. The changes in miR-146a and miR-155 after repolarization might be related to repolarization of

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human alveolar macrophages. Our data also showed that both miR-155 and miR-146a

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increased when M2 was repolarized to M1, and the changes might be transitional during repolarization. It meant that there was a close relationship between MIR - 155, MIR - 146a and macrophage repolarization. Furthermore, miR-146a inhibition resulted in increased TNF-α and IL-6 expression, while MGL-1 expression decreased after and miR-155 inhibition. Moreover, silencing of MIR-155 or MIR-146a was associated with changes in the expression of markers of M1 (TNF-α,

IL-6) and M2 (MGL-1) macrophages (Fig. 5). These observations indicate the roles of miR-146a and miR-155 in the secretion of inflammatory factors. This suggested that MIR - 155 and MIR - 146a play an important role in the regulation of alveolar macrophage repolarization. Thus, it can be speculated that repolarization of human alveolar macrophages can be stimulated in vitro by regulating the expression of miR-146a and miR-155. And we assume that the MIR - 155 and MIR - 146a may also effect the repolarization of alveolar macrophages from patients with

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pneumonia just like healthy volunteers. Furthermore, this indicates the possibility

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that pneumonia may be treated by changing the repolarization direction and the proportion of M1/M2 macrophages under miRNA-directed regulation.

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In summary, this study demonstrates that healthy human alveolar macrophages

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can be induced to repolarize rapidly in vitro. Moreover, expression of miR-155 and miR-146a are implicated in the mechanism of macrophages repolarization and secretion of inflammatory factors. The repolarization of M1/M2 alveolar macrophages through MIR - 155 and MIR - 146a regulation may be a beneficial alternative to

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antibiotics in the treatment of pneumonia. However, the corresponding process in vivo

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is highly complex and repolarization of alveolar macrophages to different subtypes is required for protection under different circumstances and periods. In addition to cytokines, other factors such as various bacteria, can also induce polarization [29]. Previous studies have shown that bacterial infection leading to pneumonia induces macrophages polarization. However, the effects of different pathogens on macrophage repolarization and the importance of each subtype at different stages of pneumonia as

well as the interactions between the subtypes remain to be elucidated. ACKNOWLEDGEMENTS Dr. Feng-Li Zhou, Dr Yu-Qi Zhou and Dr. Jing Huang were greatly appreciated for assisting in the collection of tissue and BAL samples. This work was supported by the National Nature Science Foundation of China under project No.81170004. The authors report no declarations of interest.

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Declaration of interest. The authors report no declarations of interest.

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FIG. 1 Expression of surface markers of alveolar macrophages in healthy volunteers and patients with pneumonia. Alveolar macrophages were obtained from the BALF of healthy volunteers and patients. As markers of the M1 and M2 subtypes, CD80 and CD163were detected by flow cytometry. (A) CD80 expression on alveolar macrophages of healthy volunteers and patients. (B) CD163 expression on alveolar macrophages in healthy volunteers and patients (C)The difference in surface

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markers CD80 and CD163 on alveolar macrophages between healthy volunteers and

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patients with pneumonia. Data pooled from at least two experiments.

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FIG. 2 The morphological changes of human alveolar macrophages after

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repolarization. Macrophages were cultured for 48 h in the presence of LPS (1 µg/ml) and IFNγ (10 ng/ml) or IL-4 (10 ng/ml) and IL-13 (10 ng/ml). Images show the

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morphological changes of alveolar macrophages following stimulation for 24 h (A,B) and 48 h (C,D). (E) Image of unstimulated alveolar macrophages.

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FIG. 3 Effect of polarization of macrophage on inflammatory factors (in

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healthy volunteers and patients with pneumonia) and MIRNA (in healthy volunteers). (A–D)Cells were stimulated with LPS (1 µg/ml) and IFNγ (10 ng/ml) or IL-4 (10 ng/ml) and IL-13 (10 ng/ml) to obtain M1 or M2 macrophages, respectively.

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After 48 h, the macrophages were collected and analyzed by qRT-PCR. Data represent relative expression of gene.(E) Changes in MGL1 and TNF-α expression at the RNA

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level after stimulation of macrophages from patients with pneumonia with LPS/IFN-γ (M1) or IL-4/IL-13 (M2) in macrophages from patients with pneumonia. (F) Changes in miRNA expression in alveolar macrophages from healthy volunteers after stimulation with TNF- α and IFN-γ for 24h and 48 h. Error bars represent SEMs of data pooled from at least from three experiments. *P < 0.05.

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FIG. 4 The expression of miR-155 and miR-146a in alveolar macrophages of healthy volunteers and patients with pneumonia. Error bars in this panel represent SEMs from data pooled at least from three experiments. Gene was analyzed quantitatively by qPCR.

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FIG. 5 Changes in TNF-α, IL-6 and MGL-1 in human alveolar macrophages at

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the RNA level after inhibition of MIR-155 or MIR-146a. Relative expression of gene indicate the difference between different groups. Error bars in panels A, B, and C represent SEMs from data pooled at least from three experiments. * P < 0.05, in

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comparisons between the indicated data and the healthy volunteers.

Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro.

Macrophages play an important role in inflammatory responses; however, miRNA-mediated repolarization of macrophages is essential for fulfilling this f...
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