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Exosomal miR-155-5p drives widespread macrophage M1 polarization in hypervirulent Klebsiella pneumoniae-induced acute lung injury via the MSK1/p38-MAPK axis | Cellular & Molecular Biology Letters

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Adult BALB/C mice were obtained from SiPeiFu Biotechnology (Beijing, China). All animal research protocols were approved by the Animal Experiment Ethics Committee of Fujian Medical University (Issue No.: IACUC FJMU 2023-0062). All experiments conform to all relevant regulatory standards.

Bacterial culture

HvKp strains were obtained from the laboratory’s collection. Bacteria were identified based on previously reported characteristics [20]. Bacteria were cultured in LB (Haibo, China) medium at 35 °C with shaking for 4–6 h. After that, the bacterial cultures were then centrifuged at 5000 g for 15 min to collect bacteria. Then add 10 mL PBS and resuspended pellet, followed by another centrifugation at 5000g for 15 min, and discard supernatant again. The pellet was resuspended in PBS to obtain a bacterial suspension. Bacterial concentrations were assessed by serial dilutions and quantified by measuring the optical density at 600 nm (OD600). The bacterial suspension was further diluted to achieve the desired colony-forming unit (CFU) concentrations for each experiment.

Induction of ALI mouse model with IHvKp

The suspension of hvKp was inactivated using autoclaving. Briefly, the bacterial suspension in glass vials was covered with autoclave indicator tape and subjected to a high-pressure autoclaving program at 121.3 °C for 15 min. If the indicator tape changed color after autoclaving, it indicated successful bacterial inactivation.

To establish the ALI mouse model, three male BALB/C mice were intravenously injected with iHvKp (1010 CFU/mouse), and an equal number of mice were injected with PBS as a negative control. At 24 h after injection or when humane endpoints were reached, such as decreased body temperature (lower than 30 °C), poor response to external stimuli, or inability to move freely, mice were euthanized and collected broncho-alveolar lavage fluid (BALF) and lung tissues.

Lung tissue analysis

Lung tissues were fixed in 4% paraformaldehyde for 24 h. The fixed tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for microscopic examination.

MPO assay

Myeloperoxidase (MPO) activity was measured to assess the accumulation of inflammatory cells. Lung tissues were homogenized in buffer (w/v = 1:19), and MPO activity was determined using the MPO-test kit (ZCIBIO, China).

BCA assay

The total protein concentration in the BALF was measured to assess lung permeability using a BCA assay kit (Vazyme, China), following the manufacturer’s instructions.

Cell culture

RAW264.7 (Cat. CL-0190) or HEK293T (Cat. CL-0005) cells were obtained from Procell (Wuhan, China). Cells were cultured in a specialized culture medium (Procell, China). Exosomes and cells were co-cultured using an exosome-specific medium (Umibio, China).

RAW264.7 activation was achieved by co-culturing with iHvKp. The cells were treated with a multiplicity of infection (MOI) of 100:1 (bacteria to cells) in culture dishes. After 48 h, cells and supernatants were collected. Cell activation was assessed by determining M1 polarization and inflammatory response, and the supernatant was collected for exosome isolation. The negative control supernatant was obtained by replacing iHvKp with PBS during the stimulation process.

Exosome isolation, characterization, labeling, and uptake

Exosomes were isolated using the exosome isolation and purification kit (Umibio, China), following the instructions provided. In brief, the collected cell culture supernatant was centrifuged at 3000g/min for 10 min to eliminate cells. After that, the supernatant was mixed with exosome concentration solution, vigorously shaken for 1 min, and incubated overnight at 4 °C. The mixture was then centrifuged at 10000g/min for 1 h, and the precipitate was retained and resuspended in PBS. Subsequently, another centrifugation at 12000g/min for 2 min was performed, and the resulting supernatant, passed through an exosome purification filter, yielded the exosome solution.

After isolation, exosomes were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Additionally, protein markers such as ALIX, TSG101, and CD63 were identified through Western blot analysis.

To investigate whether exosomes were taken up by cells, they were labeled with PKH67/PKH26 before injection or co-culture. The labeling was performed using the exosome PKH67/PKH26-label Kit (Umibio, China), following the provided instructions. Briefly, exosome solution (200 μl) was mixed with the staining solution (50 μl), vigorously shaken for 1 min, and incubated for 10 min. After the incubation, the excess dye was removed by exosome extraction, and the resulting solution contained PKH67-labeled exosomes. After injection or co-culture for 6 h, lung tissues or cells were fixed, stained with DAPI, and observed under a fluorescence microscope.


The exosome samples (20 μl) were carefully added drop by drop onto 200-mesh grids and incubated at room temperature for 10 min. Following this, the grids were subjected to negative staining using 2% phosphotungstic acid for 3 min. Excess liquid was removed using filter paper, and the samples were subsequently examined using an HT7800 transmission electron microscope (Hitachi, Japan).

In vivo injection and in vitro co-culture of exosomes

Quantification of isolated exosomes was performed using the BCA assay as described previously [21]. In vivo, three male BALB/C mice per group were intravenously injected with PBS-exo or iHvKp-exo (300 μg/mouse), while an equal number of mice were injected with PBS as a negative control. In vitro, RAW264.7 cells were co-cultured with PBS-exo or iHvKp-exo (50 μg/ml).

Nucleic acid transfection

Transfection of miR-155-5p inhibitor into mouse lung tissues was performed using in vivo-jetPEI (Polyplus, France). Eight male BALB/C mice per group were intravenously injected with 200 μl of a 5% glucose solution containing 6.4 reagent and 40 μg of miR-155-5p inhibitor or negative control one day before exosome or iHvKp injection.

For cell transfection of miR-155-5p inhibitor, miR-155-5p mimic, and their respective negative controls, the lipo3000 reagent kit (Invitrogen, USA) was used. The transfection mixture was prepared with a ratio of 10 pg of nucleic acid: 0.3 μl of lipo3000: 0.5 μl of P3000: 20 μl of MEM medium each well of a 48-well plate.

Immunofluorescence staining

Immunofluorescence staining was performed as previously described [22]. In brief, lung sections were subjected to an overnight incubation with either a mouse CD68 antibody (macrophage marker, Cat.GB113109, Servicebio, China) or an iNOS/Arg1 antibody (macrophage polarization marker, Cat.GB11119/ GB11285, Servicebio, China) at 4 °C. Following thorough washing, Alexa Fluor 488-conjugated (Cat.GB25303) or CY3-conjugated (Cat.G1223) secondary antibodies (Servicebio, China) were applied. The nucleus was stained with DAPI. The sections were visualized using a DMi8 fluorescence microscope (Leica, Germany).

Western blotting

Western blot analysis was performed as described previously [23]. Briefly, protein lysates of cells or exosomes were resolved on SDS-PAGE gels and then transferred onto polyvinylidene difluoride membranes for identification. GAPDH was used as an internal control. The following antibodies were used: anti-CD63 (1:500, Cat.ab216130, Abcam), anti-ALIX (1:2000, Cat.E6P9B, CST), anti-TSG101 (1:2000, Cat.ab125011, Abcam), anti-MSK1 (1:500, Cat.3489S, CST), anti-DUSP1 (1:1000, Cat.ab138265, Abcam), anti-p-p38MAPK (1:1000, Cat.4511T, CST), anti-p38MAPK (1:1000, Cat.8690S, CST), and anti-GAPDH (1:3000, Cat.92310SF, CST).

Nucleic acid extraction and identification

Total RNA extraction from cells or exosomes was performed using the Trizol method as previously described [12]. The total RNA extracted from cells was reverse transcribed using a reverse transcription kit (Vazyme, China). Quantification of RNA was carried out using an RT-PCR kit (Quanshijin, China). Reverse transcription and quantification of exosomal miRNA were performed using a specific kit (Tiangen, China). Data were normalized to the expression of GAPDH or U6. All RT-PCR analyses were performed using an ABI 7500 instrument. The primers are listed in Additional file 1: Table S1.

Dual-luciferase reporter assay

To investigate the interaction between miR-155-5p and the 3ʹ untranslated region (UTR) of the MSK1 gene, plasmid vectors containing the wild-type and mutant versions of the MSK1 3ʹ UTR with predicted miR-155-5p binding sites were constructed. These constructs were transfected into HEK293T cells. Additionally, a renilla luciferase vector was co-transfected in all transfections to monitor the efficiency of transfection. Luciferase activity was quantified as relative light units, with the average activity of the Photinus pyralis firefly luciferase normalized to the average activity of the renilla luciferase vector.

Flow cytometry

Macrophages were resuspended in PBS after centrifugation for FACS analysis. Anti-CD86 (BioLegend, Cat.105006, USA) was used according to the manufacturer’s instructions. Data were acquired using a C6 flow cytometer (BD, USA).

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 8.0) or SPSS (version 25). The data were tested for normal distribution using the Shapiro–Wilk test. Normally distributed data are presented as mean ± standard error of the mean (SEM). Two-group comparisons were analyzed using two-tailed Student’s t-test. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by post hoc tests. Survival rates between groups were compared using the Log-rank test. A p-value of less than 0.05 was considered statistically significant.

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