Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (19): 3905-3916.doi: 10.3864/j.issn.0578-1752.2023.19.016

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Effect of Porcine ECR1-Like Immune Adhesion on PAMs Capturing GFP-Escherichia coli

ZHANG Zheng1(), LING XiaoYa1, FAN KuoHai2, SUN Na1, SUN PanPan1, SUN YaoGui1, LI HongQuan1, YIN Wei1()   

  1. 1 Shanxi Key Laboratory for Modernization of TCVM/College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
    2 Shanxi Key Laboratory for Modernization of TCVM/Animal Experimental Center, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2022-11-26 Accepted:2023-08-04 Online:2023-10-01 Published:2023-10-08
  • Contact: YIN Wei

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Abstract:

【Objective】The aim of this study was to investigate whether the immune adherence function of porcine erythrocyte complement receptor type 1-like (ECR1-like) could promote porcine alveolar macrophages (PAMs) to capture sensitized genetic engineering bacteria GFP-E. coli, in order to explain the molecular mechanism of porcine erythrocyte immunity and its role in innate immunity. 【Method】The level of GFP-E. coli captured by PAMs was detected by flow cytometry, colony plate counting and RT-PCR, and the effect of porcine ECR1-like immune adherence on the capture of GFP-E. coli by PAMs was analyzed. Flow cytometry and cellular immunofluorescence technique were used to detect the changes of immune adherence function of porcine erythrocytes, and the number of porcine ECR1-like after the sensitized GFP-E. coli with ECR1-like immune adherence was removed by PAMs. 【Result】Flow cytometry showed that the average fluorescence intensity of PAMs in porcine erythrocyte adhesion group was significantly higher than that in blank control group (P<0.001), while the positive rate of PAMs cells in porcine erythrocyte adhesion group was significantly higher than that in blank control group (P<0.05). Colony smear count showed that the capture of GFP-E. coli by PAMs in erythrocyte adhesion group was significantly higher than that in blank control group (P<0.05). RT-PCR showed that the relative quantity of GFP-E. coli in PAMs of erythrocyte adhesion group was significantly higher than that of blank control group (P<0.01). Further blocking CR1-like on the surface of porcine erythrocyte, flow cytometry showed that the average fluorescence intensity of PAMs decreased to 256 301.56±9 208.85 (P<0.001), and the positive cell rate of PAMs decreased to (88.32±0.92)% (P>0.05). Colony count showed that the capture of GFP-E. coli in PAMs decreased to (136 666±8 818) CFU/mL (P<0.05), and RT-PCR showed that the relative quantity of GFP-E. coli in PAMs decreased significantly (P<0.01). Using cell flow and circulation interaction technique, it was found that the average fluorescence intensity of GFP-E. coli sensitized by porcine erythrocyte immune adherence decreased from 2 892.18±47.76 before circulation to 2 407.43±141.78 (P<0.05), and the positive cell rate decreased from (20.58±0.36)% before circulation to (17.39±0.23)% (P<0.05). The adhesion level was significantly lower than that before circulation. Meanwhile, the results of indirect immunofluorescence test showed that the average fluorescence intensity of porcine ECR1-like decreased from 344.33±37.92 before to 291.56±11.99 (P<0.05), and the positive cell rate decreased from (30.20±1.24)% before to (28.27±0.64)% (P<0.05). 【Conclusion】Porcine ECR1-like promoted the capture of sensitized GFP-E.coli by PAMs through its immune adhesion function. After PAMs removed sensitized GFP-E. coli adhered to the surface of porcine erythrocyte, the activity of CR1-like of porcine erythrocyte decreased, and the immune adhesion function decreased too.

Key words: porcine, erythrocyte, PAMs, CR1-like, immune adherence

Table 1

Primer sequences"

基因 Gene 引物序列 Primers sequence（5′-3′）
β-D-galactosidase F: CGTTATGTGATGCTGCACGG
R: CGGATCGTTCGGGTAGTGAG
GAPDH F: TTGGCTACAGCAACAGGGTG
R: CAGGAGATGCTCGGTGTGTT

Fig. 1

Flow cytometry diagram of PAMs positive cell rate and mean fluorescence intensity after blocking porcine ECR1-like activity A: Blank control group; B: Erythrocyte adhesion group; C: ECR1-like blocking group; D: Isotype control group. The same as Figs 2-5"

Table 2

Effect of blocking porcine ECR1-like activity on the rate of positive cells and mean fluorescence intensity of PAMs"

组别 Groups 阳性细胞率 Positive cell rate (%) 平均荧光强度 Mean fluorescence intensity
A（空白对照组Blank cell group） 86.49±1.53 207488.60±1405.65
B（红细胞黏附组Erythrocyte adhesion group） 90.74±1.07 297655.94±14833.96
C（ECR1-like阻断组ECR1-like blocking group） 88.32±0.92 256301.56±9208.85
D（同型对照组Isotype control group） 91.82±3.12 300211.32±8001.76

Fig. 2

One-way ANOVA results of positive cell rate and average fluorescence intensity of PAMs"

Fig. 3

Plate coating of GFP-E. coli captured by PAMs after blocking porcine ECR1-like activity"

Table 3

Effects of blocking porcine ECR1-like activity on the results of GFP-E. coli colony counting captured by PAMs"

组别 Groups CFU (mL)
A（空白对照组Blank cell group） 111666±25927
B（红细胞黏附组Erythrocyte adhesion group） 203333±4713
C（ECR1-like阻断组ECR1-like blocking group） 136666±8818
D（同型对照组Isotype control group） 186666±28284

Fig. 4

One-way ANOVA results of PAMs capture GFP-E. coli"

Fig. 5

One-way ANOVA results of the relative quantity of GFP-E coli captured by PAMs"

Fig. 6

Flow cytometry diagram of the detection of porcine red blood cell immune adhesion levels before and after PAMs removal of sensitized GFP-E. coli Ⅰ: Blank control group, Ⅱ: Test group, Ⅲ: Negative control group. The same as Fig. 7 and Fig. 8"

Table 4

Immunoadhesion positive cell rate and mean fluorescence intensity of porcine erythrocytes before and after removal of PAMs to sensitize GFP-E. coli"

组别
Groups		 阳性细胞率
Positive cell rate (%)		 平均荧光强度
Mean fluorescence intensity
Ⅰ（空白对照组
Blank control group）		 循环前免疫黏附 Pre-circulating immune adhesion 20.06±0.44 2846.64±26.84
循环后免疫黏附 Post-circulatory immune adhesion 19.07±1.32 2729.63±49.82
Ⅱ（试验组
Test group）		 循环前免疫黏附 Pre-circulating immune adhesion 20.58±0.36 2892.18±47.76
循环后免疫黏附 Post-circulatory immune adhesion 17.39±0.23 2407.43±141.78
Ⅲ（阴性对照组
Negative control group）		 循环前免疫黏附Pre-circulating immune adhesion 20.83±0.23 2835.38±18.99
循环后免疫黏附 Post-circulatory immune adhesion 21.25±0.01 2877.65±19.61

Fig. 7

Comparison of the immune adhesion level (mean fluorescence intensity) of porcine erythrocytes before and after PAMs removal of sensitized GFP-E. coli"

Fig. 8

Comparison of the immune adhesion level (positive cell rate) of porcine erythrocytes before and after PAMs removal of sensitized GFP-E. coli"

Fig. 9

Flow cytometry diagram of porcine ECR1-like quantity level detection before and after removal of sensitized GFP-E. coli by PAMs a: Normal control group, b: Treatment group Ⅰ, c: Treatment group Ⅱ, d: Treatment group Ⅲ, e: Isotype control group, f: Blank cell group. The same as Fig. 10"

Table 5

Porcine ECR1-like positive cell rate and average fluorescence intensity before and after removal of PAMs to sensitize GFP-E. coli"

组别 Groups 阳性细胞率 Positive cell rate (%) 平均荧光强度 Mean fluorescence intensity
a（正常对照组Normal control group） 30.20±1.24 344.33±37.92
b（Ⅰ处理组Treatment group Ⅰ） 29.79±0.21 333.58±15.60
c（Ⅱ处理组Treatment group Ⅱ） 28.27±0.64 291.56±11.99
d（Ⅲ处理组Treatment group Ⅲ） 30.07±1.15 341.49±34.80
e（同型对照组Isotype control group） 15.27±1.91 141.10±10.30
f（空白细胞组Blank cell group） / 37.88±0.33

Fig. 10

Comparison of ECR1-like quantity in porcine after removal of sensitized GFP-E. coli by PAMs"

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