In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages

Animal ethics

All animals used in the present experiments were cared for humanely and the use of the animals was approved by the ethics committee at the Animal Science and Veterinary Medicine College of Shanxi Agriculture University in China (No. SXAU-ASVM-16019). All experiments were conducted in compliance with the International Guiding Principles for Biomedical Research Involving Animals (CIOMS and ICLAS, December 2012).

Experimental animals and bacterial strains

Three Healthy Landrace pig, weighing 20 ± 2 kg, were purchased from Xinsihai pig farm in Wuxiang County, Shanxi Province, China. Healthy rabbits, weighing 2 ± 0.5 kg, were purchased from Shanxi Agricultural University Animal Production Laboratory. Wild type Escherichia coli (WT E. coli) cultured and then freeze-stored in our laboratory were used, The bacterial concentration measured by plate counting method was 4.846 × 105/mL.

Main reagents and equipment

The main reagents include Indian ink (0.1%, Nanjing SenBeiJia Biological Technology Co., Ltd., Nanjing, China) mouse anti-porcine CR1-like monoclonal antibody (IgG2a; previously developed in our laboratory; FITC-goat anti-mouse IgG (Abbkine Inc., California, USA); mouse IgG1 isotype antibody (Beijing Protein Innovation Co. Ltd., Beijing, China); penicillin, streptomycin, Hank’s Buffer, phosphate-buffered saline (PBS, pH = 7.0), and Trypan blue solution (Solarbio Life Sciences Co. Ltd., Beijing, China; FITC and DMSO Sigma Inc., Missouri, USA); tryptone and yeast extract (Sangon Biotech Co. Ltd., Shanghai, China); erythrocyte lysis buffer (Tiangen Biotech Co. Ltd., Beijing, China); and BSA (Jackson ImmunoResearch Inc., rural Pennsylvania, USA). The main equipment used included: BD FACScalibur flow cytometer (BD Bioscience, New York, USA), and IX81 inverted fluorescence microscope and FV1000 laser confocal microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).

Preparation of porcine erythrocyte suspension

Blood was collected aseptically from the anterior vena cava from the experimental pigs, and lymphocyte separation medium was used to isolate porcine erythrocytes. These erythrocytes were resuspended using 0.5% IgG-free BSA-Hank’s buffer and were observed and counted under a normal light microscope. The cell density was adjusted to 2.47 × 10⁷/mL for use in further experiments.

Isolation of porcine alveolar macrophages and primary culture

Pigs were anesthetized with ketamine and their lungs were obtained by aseptic surgery, Phosphate buffer saline (PBS) was used to flush the lung surface to remove contaminants. Then, 100 mL PBS (pH 7.4) was aspirated and injected into the lungs through the trachea, and the lavage fluid was collected and centrifuged at 112g for 5 min. The supernatant was discarded, and the cells were resuspended in RPMI 1,640 medium. This procedure was repeated twice to obtain porcine alveolar macrophages (PAMs). The PAM suspension was pipetted into a 1.5 mL centrifuge Tube and centrifuged at 112g for 5 min. The supernatant was discarded and the cells were resuspended in 12 mL of 1,640 cell culture medium. The cells were then seeded in six-well plates at a density of 2 × 106 cells/well. After incubation in a cell culture incubator for 12 h, the plates were removed and the culture medium was discarded. PBS was used to gently wash the plates and fresh 1,640 cell culture media was added at two mL/well. The plates were subsequently placed in a cell culture incubator and the cell culture medium was changed once every 24 h.

PAM survival rate and phagocytosis rate

Porcine alveolar macrophage suspension (100 μL) was mixed with an equal volume of 0.4% Trypan blue. After staining for 2–3 min, the stained and unstained cells were counted to calculate the survival rate. The PAM suspension was pipetted and seeded at a density of 1 × 10⁵/mL in 24-well plates. After culturing for 2 h in a cell culture incubator, 100 μL of ink (autoclaved, diluted 10× in PBS) was added to each well while an equal volume of PBS was added to the control group. Each experiment was performed in triplicates.

FITC labeling of WT E. coli

FITC powder (two mg) was collected in the dark and dissolved in 10 mL of PBS (pH 8.4). Then, 500 μL of this FITC solution was added to one mL of WT E. coli suspension. The mixture was incubated in a shaking incubator at 37 °C, 220 r/min for 1 h in the dark. After incubation, the solution was centrifuged at 2,795 g for 5 min, and the supernatant was discarded. The bacterial pellet was resuspended in one mL of 0.5% BSA-Hank’s buffer; this step was repeated twice. The fluorescence-quenching rate of the resuspended fluorescent bacteria was calculated at time points of 1 h, 1.5 h, 2 h, 2 h 5 min, 2 h 10 min, 2 h 15 min, 2 h 30 min, 2 h 45 min, and 3 h.

Preparation of oposinized E. coli

Blood (two mL) was collected from the hearts of the experimental rabbits in ordinary blood collection tubes with no anticoagulant. These tubes were placed in a water bath, maintained at 37 °C, for 30 min and then centrifuged at 2,795g for 15 min. The serum thus obtained was collected for further use. Then, 50 μL of rabbit serum was added to 200 μL FITC-labeled WT E. coli suspension. The mixture was incubated in the dark in a water bath at 37 °C for 30 min and centrifuged at 2,795g for 5 min. The supernatant was discarded, and 200 μL of 0.5% BSA-Hank’s solution was used to resuspend the pellet. This procedure was repeated twice and the bacteria were observed under the microscope for any abnormalities before use.

Detection of competitive binding of PAM to oposinized WT E. coli

A total of 200 μL of porcine erythrocyte suspension and 200 μL of the oposinized E. coli-FITC suspension were mixed and incubated at 37 °C in the dark for 30 min. The mixture was then centrifuged, and the pellet was washed twice and resuspended. Two milliliters of PAM solution was added to sterile six-well plates and incubated for 15 min. Then, the prepared cell suspension was added and the plates were incubated at 37 °C for 2 h. The cells were then harvested and 600 μL of erythrocyte lysis buffer was added. The suspension was centrifuged and washed twice. The pellet was resuspended in 300 μL of 1% paraformaldehyde fixing solution. The average fluorescence intensity of PAM was measured by flow cytometry. Statistical analysis of average fluorescence intensity of samples was carried out.

Detection of CR1-like on porcine erythrocytes during transfer of oposinized E. coli

The oposinized E. coli suspension, porcine erythrocyte suspension, and PAM suspension was prepared as described in the previous sections. Five sterile 1.5 mL centrifuge Tubes were labeled as groups I, II, III, IV, and V. In groups I–IV, 100 μL of porcine erythrocyte suspension was added. In group I, 100 μL of oposinized E. coli suspension was added, incubated for 30 min, centrifuged, and washed twice. The bacteria were then resuspended in 100 μL of PAM suspension and mixed evenly. In group II, only 100 μL of PAM suspension was added and mixed evenly. Groups I and II were incubated for 2 h; erythrocytes were collected and sequentially incubated in the dark at 37 °C with mouse anti-porcine CR1-like monoclonal antibody and FITC-goat anti mouse IgG2. Groups III–V were control groups for flow cytometry: Group III had anti-porcine CR1-like monoclonal antibody, and FITC-goat anti mouse IgG2 added sequentially as described earlier. In group IV, mouse isotype control antibodies were added at an antibody dilution of 1:200, according to the instruction manual. In group V, no cells were added, but PBS was added as a control. The above groups were loaded in flow cytometry tubes and sent to Shanxi Medical University for quantitation of the average fluorescence intensity of erythrocytes (Table 1).

Ink phagocytosis test result

Porcine alveolar macrophages obtained after isolation were allowed to revive and were cultured. Inverted microscopy observations showed that most of the cells were circular, morphologically uniform, the PAM have phagocytic function, and were in a semi-adherent state. The survival rate of cells calculated after Trypan blue staining was 97.2% (Fig. 1A). Ink was added to the PAM culture system for continuing culture. Inverted microscopy observations of the ink-stained cells showed the phagocytosed particles inside the cells, and the PAM phagocytosis rate was calculated to be 98.8% (Fig. 1B).

WT E. coli were successfully labeled with FITC

After WT E. coli were labeled with FITC dye, they were placed under a direct microscope. The bacteria appeared as short rods with size of one to two μm. Under the fluorescence view, WT E. coli presented a strong green fluorescence, demonstrating the successful labeling of bacteria; 92.3% bacteria were fluorescently labeled (Fig. 2). The bacteria were resuspended in PBS buffer and transferred to a laser confocal microscope to observe the fluorescence quenching time. Under a fluorescence field, the green fluorescence of the bacteria was gradually quenched from 0 to 3.4 h. The quenching rates were 3.7%, 10.9%, 11.2%, 11.7%, 14.6%, and 41.9%, at 1.5 h, 2 h, 2 h 15 min, 2 h 30 min, 3 h, and 3 h 45 min, respectively.

WT-E. coli-FITC were bound and internalized by PAMs

E. coli oposinized by fresh rabbit serum was first incubated with erythrocytes and then co-incubated with PAMs, and subjected to laser confocal microscopy to observe the transfer process. The fluorescent bacteria were attached to the surface of porcine erythrocytes or PAMs, and there were no considerable changes in the adhesion status of fluorescent bacteria from 0 to 2 h. At 2 h 5 min, 7 o’clock direction on a porcine erythrocyte had three fluorescent bacteria attached to it, and this erythrocyte was located at the junction of three PAMs (Fig. 3A). At 2 h 11 min, the fluorescence at 11 o’clock direction became slightly weaker and the fluorescence became clearer after focusing. However, the contours of porcine erythrocytes showed a blurring phenomenon (Fig. 3B). At 2 h 23 min, 2 h 27 min, and 2 h 36 min, bacterial fluorescence intensity weakened gradually (Figs. 3C–3E). At 2 h 59 min, no fluorescent bacteria were seen at the 7 o’clock direction on the porcine erythrocyte. Thus, as the observation time increases, the weakening of the fluorescence intensity of the bacteria is not due to the quenching of fluorescence but is caused by the movement of fluorescent bacteria from their original adhesion location to the junction between erythrocytes and PAMs.

Competitive binding of PAM to fluorescent E. coli

After erythrocyte lysis in the above co-incubation system, the samples were subjected to flow cytometry. The mean fluorescence intensity of PAMs in the co-incubation group was 638.63 ± 44.82 (Fig. 4A) whereas the mean fluorescence intensity of PAMs in the control group was 5.34 ± 0.08 (Fig. 4B); thus, the mean fluorescence intensity of PAMs in the co-incubation group was significantly higher than the control group (P < 0.01) (Fig. 4C).

Level of CR1-like on porcine erythrocytes during the transfer of oposinized WT E. coli

The fluorescence intensity of porcine erythrocytes as detected by flow cytometry for groups I–V was 70.78, 104.56, 111.04, 4.39, and 2.77, respectively (Figs. 5A–5E). The fluorescence intensities of porcine erythrocytes in groups I–III were significantly higher than those in groups IV and V (P < 0.05). The fluorescence intensity of porcine erythrocytes in group I was significantly lower than that of groups II and III (P < 0.05) whereas the fluorescence intensity of porcine erythrocytes in group II was lower than that in group III, but this difference was not statistically significant (P > 0.05) (Fig. 5F).

Thus, there were no significant changes in CR1-like expression levels of porcine erythrocytes between porcine erythrocytes alone and those co-incubated with PAMs. However, when porcine erythrocytes, fluorescent bacteria and PAMs were co-incubated, the expression levels of CR1-like on porcine erythrocytes decreased.