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

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.

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.

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).

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.