High expression of miR-222-3p in children with Mycoplasma pneumoniae pneumonia

36 children with MPP from Children’s Hospital of Soochow University were enrolled in this study from March 2014 to June 2015, and immunodeficiency, premature delivery, recurrent pneumonia and recent use of immunosuppressive agents and immunomodulators were excluded.There were twenty males and sixteen females, with an average age of (6.74 ± 2.82) years. Among them, sixteen children with pleural effusion. Twenty-seven healthy children in the same period served as control group, fifteen males and twelve females, with an average age of (7.08 ± 2.74) years. There was no difference in sex and age between the two groups (p <  0.05), which was comparable.The inclusion criteria of the control group were no history of immune system diseases and chronic infectious diseases and no respiratory tract infections, other acute and chronic diseases, no allergies and diseases closely related to immunity in the past two weeks. Ethical approval for the study was received from the Institutional Medical Ethics Review Board of Children’s Hospital of Soochow University, and the study was performed in accordance with the Declaration of Helsinki. The diagnosis of M. pneumoniae infection was based on serologic testing and confirmed by polymerase chain reaction (PCR) tests of nasopharyngeal secretions. Diagnosis in all patients was made based on clinical and radiological signs of CAP in all patients according to British Thoracic Society Guidelines for the Management of Community Acquired Pneumonia [7]. Upon patient admission to the hospital, their pediatricians completed a questionnaire regarding the patient demographic and clinical data. Meanwhile, peripheral blood samples were obtained for use in miR-222-3p and CD4 detection. Twenty-seven of age-matched controls who had not experienced any acute respiratory infections within the previous four weeks were chosen randomly from surgery wards.

Peripheral blood was collected for laboratory examination and for miR-222-3p and CD4 mRNA detection. Laboratory data were also collected, such as white blood cell count (WBC), absolute neutrophil count, C-reactive protein (CRP), lactate dehydrogenase (LDH), and lymphocytes subgroups.

Peripheral blood samples from three healthy controls and three MPP cases were obtained and then drawn into EDTA-containing glass tubes. Samples were immediately centrifuged at 3500×g for 15 min at 4 °C. The resulting plasma samples were then stored at − 80 °C until analysis. To determine the levels of free haemoglobin in the samples, 1.5 μl of total plasma was analyzed spectrophotometrically (NanoDrop 1000, Thermo Scientific) [8]. Absorbance levels above 0.2 at 414 nm were indicative of free haemoglobin and thereby a higher degree of haemolysis and such samples were excluded from further analysis.

Total RNA was isolated from human plasma [9]. A 200-μl sample of the plasma was transferred into a 1.5 ml tube and mixed with 750 μl of lysis mixture containing RNA lysis reagent (Trizol, Ambion, USA). Microarray hybridization, data generation, and normalization were performed by Kangchen Biological Engineering Co. Ltd. Using a human miRNA chip (miRCURY™, Exiqon, Denmark), which contains probes for 3100 miRNAs. Normalization was performed using quantile algorithm. MicroRNAs were considered to be differentially expressed if they produced a p-value of <  0.05 and a false discovery rate of ≤0.05. Differentially expressed miRNAs were also designated as up-regulated or down-regulated if the differrence in MPP children compared to controls was more than two-fold.

Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Next, 1 μg of total RNA was subjected to reverse transcription PCR using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) according to manufacturer’s protocol. The thermocycling conditions were: 60 min at 42 °C, followed by 15 min at 70 °C, and 30 min at 4 °C. qRT-PCR was performed using a TaqMan Universal PCR Master Mix kit (Applied Biosystems) in an Bio-Rad iQ5 Real-Time PCR System and U6 or β-action was used as an endogenous control for miR-222-3p and CD4 respectively. Reactions were performed in triplicate. The thermocycling conditions for amplifying miR-222-3p were: 95 °C for 30 s, followed by 45 cycles of 5 s at 95 °C and 60 °C for 30 s, and concluded with 1 min at 60 °C. Meanwhile, the thermocycling conditions for amplifying CD4 were: 95 °C for 30 s, followed by 45 cycles of 5 s at 95 °C and 57 °C for 20 s, and concluded with 1 min at 60 °C [10]. After finalization of the qRT-PCR experiments, the average values of the cycle threshold (Ct) of the reactions in triplicate were determined. Data analysis was performedusing the 2^-ΔΔCt method.

THP-1 cells with a density of 1 × 10⁵ cells/ml were cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) and then harvested and centrifuged after reaching exponential growth. The resulting cell pellet was suspended in serum-free medium, 1 × 10⁶ cells were distributed across a 24-well plate. Different concentrations of LAMPs (2 μg/ml, 4 μg/ml, 6 μg/ml) were added into the wells and the THP-1 cells were co-culture with LAMPs for 16 h; a control group was treated with phosphate buffered saline(PBS) instead of LAMPs. Then THP-1 cells were then harvested for miR-222-3p and CD4 detection by real-time PCR as described above.

Eight-week-old male BABL/c mice were purchased from the Chinese Academy of Sciences. All animal studies were approved by the institutional animal care and use committee at Soochow University and complied with the animal welfare act. The methods applied in this study were carried out in accordance with the approved guidelines. Briefly, mice were randomly divided into two groups (n = 5 per group): 1) control group treated with saline, 2) LAMP group stimulated by LAMPs. Each mouse was intranasal instillated with 50 μg of LAMPs or saline. The experiment was terminated at 48 h after LAMPs inhalation, and the lung tissues were harvested thereafter. This in vivo study was carried out in two separate experiments. BALF samples and lung tissues were harvested using a previously described method [11]. The collected fluid was then centrifuged at 500×g for 2 min at 4 °C. Trizol was added to the resulting pellet, which was then stored at − 80 °C, then harvested for miR-222-3p and CD4 detection by real-time PCR as described above. Meanwhile, the lung was fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Lung sections of 4 μm in size were stained with hematoxylin and eosin for morphological evaluation.

A total of 36 cases with MPP and 27 control children with selective surgery were enrolled in the present study. As shown in Table 1, the amounts of absolute neutrophils, CRP, LDH, CD3⁻CD16⁺CD56⁺ NK cells and CD19⁺CD23⁺ B cells in children with MPP were all significantly increased compared with controls (all p <  0.05).

A heat map was generated from the microarray analysis of plasma from control children and those with MPP. Compared with the controls, there were 26 differentially expressed miRNAs with differences of > 2-fold in the plasma from children with MPP; Of these 19 miRNAs were up-regulated and 7 miRNAs were down-regulated (Fig. 1).

According to the Target Scan 7.1 database, CD4 is one of the direct targets of miR-222-3p. Children with MPP had significantly higher levels of miR-222-3p in tneir peripheral blood mononuclear cells compared with controls (1.5 ± 0.2 vs. 1.0 ± 0.2, relative expression) and lower levels of CD4 mRNA (0.5 ± 0.2 vs. 1.0 ± 0.4, relative expression)(Fig. 2). Meanwhile, MPP cases with pleural effusion had higher levels of miR-222-3p compared with those that lacked pleural effusion; but no difference in CD4 mRNA levels was found between these groups(Fig. 2). No correlation was found between miR-222-3p and CD4 expression (r = − 0.231, p = 0.175).

Macrophages play a vital role in excessive inflammation caused by M. pneumoniae infection [12]. Based on this, we stimulated THP-1 cells with LAMPs from M. pneumoniae to explore the in vitro expression of miR-222-3p and CD4 mRNA in M. pneumoniae-stimulated monocyte cells. The level of miR-222-3p expressed by THP-1 cells increased in a dose-dependent manner after stimulation with LAMPs (Fig. 3). Furthmore, the miR-222-3p level was positively associated with the LAMP concentration (Fig. 4). In contrast, the CD4 level in THP-1 cells decreased in a dose-dependent manner after LAMP stimulation.

To determine the in vivo expression of miR-222-3p in BALF, LAMPs from M. pneumoniae were used to stimulate the lungs of BALB/c mice. Massive infiltrations of inflammatory cells were found around the bronchioles in LAMP-stimulated mice compared with controls (Fig. 5). Furthermore the level of miR-222-3p in the BALF was significantly higher for LAMP-stimulated mice than for saline-treated controls (Fig. 5) .