General condition of mice
With the prolongation of the feeding time, the weight of the two groups of mice showed an increasing trend, but the increase in the experimental group was more significant (Table 2). The mice of control group showed no abnormal changes in the liver and abdominal cavity after dissection. The liver texture was soft, smooth, ruddy in color without obvious adhesion. The anatomical position was normal. After dissecting the mice in the experimental group, it was found that as the infection continued, the liver of the mice gradually hardened, the color gradually faded, and the anatomical position gradually deviated from the normal anatomical position. At 30d after infection, the vesicles were seen on the liver, then the vesicles gradually increased. The boundary between vesicles and tissue was blurred. The vesicles were also formed in the abdominal cavity. At the late infection, abdominal cavity was almost full of vesicles (Fig.1, Fig.2).
Table 2 Changes in the weight of mice infected with Echinococcus multilocularis in different periods(͞x ± s)
Days after infection (d)
|
n
|
Weight (g)
|
Control group
|
Experimental group
|
2
|
8
|
21.52±0.65
|
21.09±1.01
|
8
|
8
|
21.82±0.77
|
21.93±0.85
|
30
|
8
|
23.65±0.58
|
24.71±1.03
|
90
|
8
|
24.88±0.44
|
30.23±2.39*
|
180
|
8
|
30.46±1.00
|
37.56±3.36**
|
Note: The experimental group was compared with the control group at the same period * P<0.05, ** P<0.01
Histological analysis of the liver tissue
The control group’s mice in all periods had normal liver tissue structure and complete hepatic lobules. Hepatocytes were arranged neatly with clear and complete borders. There was no edema, necrosis, and ballooning degeneration in liver cell. After observing the mice in experimental group, we found that as the infection continued, the hepatocytes gradually developed from initial edema to ballooning degeneration and steatosis. At 8d of infection, the mild inflammation in the portal area began to appear, and it became worse with the prolongation of the infection time. At the same time, a small number of focal necrosis could be seen in the hepatic lobules. With the prolongation of infection time, it gradually increased, developed into fusion necrosis or bridging necrosis and involved many hepatic lobules. In the late stage of infection, a large number of fibroblasts around the lesion proliferated to form a fibrous layer, and a large number of inflammatory cells infiltrated. Normal hepatic lobule structures were no longer visible (Fig.3).
According to the pathological changes in HE stained hepatic lobules of liver tissue of mice infected with Echinococcus multilocularis, lesions in the manifold area, other abnormal structures and substances, combined with chronic hepatitis GS classification, a score scale for the degree of Echinococcus multilocularis infection in mice (Table 3). Tissue samples scored according to Table 3. After summing up the scores, the higher the score, the severer the infection. (Fig.4).
Table 3 Liver infection of Echinococcus multilocularis severity score (mouse)
Degree of inflammation
|
Vesicles*(number)
|
Abnormal structures and substances
|
Score
|
Hepatic lobule
|
Portal area and surrounding
|
No inflammation
|
No inflammation
|
0
|
No obvious change
|
0
|
Degeneration, small amount spotted and focal necrosis
|
Mild inflammation (Inflammatory cells occupy less than 1/3 of the portal area)
|
1-3
|
Granuloma
|
1
|
Degeneration, large amount spotted and focal necrosis
|
|
4-6
|
|
2
|
Confluent necrosis or bridging necrosis
|
Moderate inflammation (Inflammatory cells occupy 1/3~2/3 of the portal area)
|
7-9
|
Laminated layer and germinal layer
|
3
|
Extensive bridging of necrosis and involved many hepatic lobules
|
Severe inflammation (Inflammatory cells occupy more than 2/3 of the portal area)
|
≥10
|
Brood capsule and protoscolex
|
4
|
* Note: the average number of vesicle counts was observed in at least 3 different fields under low power microscope (magnification, ×100).
Detection results of monocyte subsets in peripheral blood
Ly6C is currently the most ideal marker for distinguishing monocyte subpopulations in mice[12]. Then we used flow cytometry to detect the proportion of monocyte subsets in the peripheral blood of mice in control and experimental groups in different periods. We found that a little change in the proportion of two kinds of monocytes in mice of control group. In experimental group, the proportion of Ly6Chi type monocytes gradually increased in the early stage of infection, reached a peak at 8d after infection. Then it began to show a downward trend. At the later stage of infection, it had been significantly lower than the control group at the same time (Fig.5 G). For Ly6Clo type monocytes, its proportion rapidly decreases to a minimum after a brief rise in the early stages of infection. Then its proportion began to rise, and at the later stage of the infection, its proportion was already significantly larger than that of the control group during the same period (Fig.5 H). The above results showed that in the continuous infection of Echinococcus multilocularis, the mainly monocyte subsets in peripheral blood transited from Ly6Chi to Ly6Clo.
Immunohistochemical results of liver tissue of Echinococcus multilocularis infection mice
Earlier, we have observed changes in the proportion of peripheral blood mononuclear cell subsets in infected mice. So did these cells reach the lesion? Subsequently, we used immunohistochemistry to detect monocyte markers, CD11b, related chemokines and their receptors CX3CL1 and CX3CR1, different types of macrophages markers, iNOS and CD163. The results showed that all indicators were low and stable in the control group. The expression of chemokine CX3CL1 increased with the prolongation of the infection time. Although the expression level decreased in the late stage of infection, it remained at a high level (Fig.7 A). Most of its expression locations were concentrated in inflammatory cell clusters or around lesions. CX3CR1 had a higher expression level than control group at the early stage of infection. Although the expression level decreased briefly at 30d after infection, it was still significantly higher than that of control group in the later period (Fig.7 B). The location of its expression gradually concentrated around the lesion with the prolongation of the infection time. For CD11b, its expression increased with the prolongation of infection time (Fig.7 C), and almost all of the expression sites were at the inflammatory cell aggregation site. The expression of iNOS that one of the markers of M1 macrophages, increased with the prolongation of the infection time, but the expression level in the late stage of infection was no longer than the intermediate stage (Fig.7 D). CD163 is one of the markers of M2 macrophages. And its expression also increased with the prolongation of infection time. The expression of CD163 in the late stage of infection was the highest during the entire infection process which is unlike iNOS (Fig.6, Fig.7 E). In summary, our results illustrate the following conclusion, under the role of chemokines and their receptors, monocytes expressing CD11b were recruited into the liver and concentrated around the lesion at the late stage of infection. Macrophages around the lesion were predominantly M1 and M2 in the early and late stages of infection, respectively.
Detection of different subsets macrophage related genes
After that, in order to verify the previous conclusions, we carried out the detections of liver macrophage-related gene. The results of qRT-PCR showed that TNF-α, a marker gene of M1 macrophages, began to increase significantly at 30d after infection. Its expression reached its peak at 90d. Although it decreased in the late period, it was still significantly higher than the control group in the same period (Fig.8 A). Arg1, one of the marker genes of M2 macrophages, also began to increase significantly at 30d after infection. But the difference was that it reached a peak in the late stage of infection with the prolongation of the infection time (Fig.8 B). The experimental results supported our previous conclusions that macrophages in the liver underwent a continuous transition from M1 to M2 in the continuous infection of Echinococcus multilocularis. So, how do these macrophages that have been polarized participate in the mice's immune response at different stages of continuous infection in Echinococcus multilocularis?
Results of detection of macrophages related cytokines
In order to explore the immune function of macrophages that have been polarized at different stages of infection, we detected several cytokines associated with different types of macrophages. First, we detected changes in the expression of IFN-γ in peripheral blood. Its expression trend was similar to that of M1-type macrophages (Fig.9 A). Both of them increase with the duration of infection, and begin to decline in the later stages of infection. Then, we detected changes in the expression of IL-17. During the entire infection process, its expression continued to increase with the prolongation of the infection time, reached a peak in the late stage (Fig.9 B). Finally, we continuously detected two cytokines, IL-4 and IL-10, and their expression trends were similar. When the infection progressed to 90d, their expression reached a peak and gradually decreased in the late stage of infection. The difference was that IL-10 was significantly higher than the control group at 30d after infection, but IL-4 was not (Fig.9 C, D). This was the difference between the two cytokines. Our results indicate that in the early stage of persistent infection with Echinococcus multilocularis infection, Th1 immune response represented by IFN-γ may play a major and important role. In the late stage of infection, maybe Th2 cytokines with higher expression were playing a major role. Therefore, no matter in the early or late stage of infection, macrophages of different subsets participated in the mice's immune regulation by synthesizing and secreting relevant cytokines, and played an indispensable role.