| The innate immune system is the first line of defense against pathogens in mammals,which has an important interaction with the living environment of animals.Ecological immunology aims to address the complexity of immune responses of wildlife in ecological and evolutionary environments,understand the differences in individual immune function,and determine the adaptive consequences of such differences.The plateau zokor?Eospalax baileyi?is an indigenous rodent on the Tibetan Plateau,which spends almost their lifetime in the underground tunnel.In order to cope with the extreme environment of hypoxia and high carbon dioxide in the tunnel,the morphological,physiological and behavioral adaptive characteristics of plateau zokor have been widely reported.Heretofore,no systematic study has been reported on the plateau zokor ecological immunity.In the present study,we investigated the innate immune indicators in the blood heterogeneity,intestinal mucosal innate immunity level and the impact of intestinal microbial diversity on its innate immune system,TLR-mediated signal transduction pathway in the spleen,and the mechanism of apoptosis and pyrolysis of peritoneal macrophages in the plateau zokor compared with low-altitude underground rodent bamboo rat?Rhizomys pruinosus?and SD rat?Rattus norvegicus?using physiological,biochemical and molecular biological methods.The objective of the present study was to explore the relationship between the habitat characteristics of the three species and the innate immune response,and reveal the innate immunity function of the plateau zokor in extreme habitats.The results showed that the bactericidal activity of whole blood in plateau zokor to Escherichia coli was significantly higher than that in bamboo rat and SD rat.However,the bactericidal activity of whole blood of plateau zokor to Staphylococcus aureus and Candida albicans were significantly lower than that of bamboo and SD rat.After LPS immune stress,the Lymph and Gran counts of plateau zokor were lower than those of bamboo rat and SD rat in the same treatment group.Plasma C3concentration of plateau zokor in the saline group was significantly higher than that of bamboo rat in the same group,and C4 concentration was significantly higher than that of the SD rat in the same group.Plasma C3 and C4 concentrations of plateau zokor in the immune stress group were significantly higher than those of bamboo and SD rats.Plasma LZM and MBL contents of plateau zokor in both saline and immune stress groups were lower than the SD rats of the same groups.The phagocytosis rate of neutrophils in plateau zokor from both groups was lower than that in the SD rat.The level of ROS in the saline group of plateau zokor was higher than that in bamboo and SD rat,but the level of ROS in the immune stress group was significantly lower than that in the SD rat.Furthermore,the plasma TCA of plateau zokor increased by 29.71%after LPS immune stress,which was significantly higher than that of the SD rat.Meanwhile,the plasma CORT content of plateau zokor was higher than that of the SD rat of the same group.The above-mentioned test innate immune indicators were lower in plateau zokor than those in bamboo rat and SD rat.This may be due to the long-term evolution and environmental stress,which makes the plateau zokor invest fewer trade-off strategies for innate immunity.We compared the morphology of the small intestine in three species.Plateau zokor has smaller the thickness of the mucosal layer of the small intestine,the length of the villi of the small intestine,and fewer goblet cells in the small intestinal epithelial cells than the bamboo rat.The number of Paneth cells and microvilli was less in plateau zokor than that in the SD rat.The content of?-DF in the small intestine of plateau zokor was significantly lower than that of SD rat,and?-DF was significantly lower than that of bamboo rat and SD rat;the activity of LZM was significantly lower than that of bamboo rat and SD rat,the activity of ACP and AKP was lower significantly than that of bamboo rat and SD rat.Also,the activities of T-NOS,i-NOS and GSH-Px in the small intestine of plateau zokor were significantly higher than those of bamboo rat and SD rat,and the activities of T-AOC and T-SOD were significantly higher than those of SD rat.The third generation and high-throughput sequencing technology of 16S full-length was used to analyze the diversity of cecal bacteria in three species,the results showed that the intestinal bacterial diversity of plateau zokor was significantly higher than that of bamboo rat,among which the relative abundance of anaerobic bacteria,Gram-negative bacteria,biofilm formation and mobile element bacteria were lower than that of other two species,while the bacterial abundance of plateau zokor was higher than that of other two species.Moreover,the relative abundance of bacteria in the intestinal stress tolerance of plateau zokor was higher,which indicated that the intestinal tract of plateau zokor had strong antioxidant stress ability in the environment of low oxygen in the tunnel,which was consistent with previous results of the small intestine.The second-generation high-throughput sequencing technology was used to analyze the diversity of the conserved regions of cecal fungi in three species,the results showed that the diversity of intestinal fungi in plateau zokor was significantly higher than that in bamboo rat and SD rat.The dominant fungal communities in the intestine of plateau zokor are Ascomycetes and Basidiomycetes,and the characteristic difference fungi are Mortierella alpina,which may weaken the innate immune activation level.These results showed that the investment of plateau zokor on innate immunity of intestinal mucosa is less and the level is lower than the other two species may be due to the environmental pressure,such as hypoxia and resource limitation.By calculating the spleen indices of the three species,we found that the spleen index of the plateau zokor was significantly lower than that of the bamboo rat and SD rat.As shown by morphological comparisons of spleens,the splenic red pulp distribution area was larger in the plateau zokor those in the bamboo rat and SD rat,while the white pulp distribution area was smaller in the plateau zokor than in the bamboo rat and SD rat.Furthermore,the trabecula was shorter and thicker in the plateau zokor than in the bamboo rat and SD rat.As shown by TEM,the proportion of immune cells in the spleen of the plateau zokor was smaller than that of the SD rat,and spleen cells exhibited a less rounded morphology in plateau zokor than those in SD rat.After LPS immune stress,the expression of TLR2 in the spleen of plateau zokor was significantly higher than that in bamboo rat and SD rat,and it was mainly expressed in the red pulp of the spleen;in contrast,the expression of TLR4 was significantly lower than that in SD rat;the expression of HIF-1?was significantly higher than that of bamboo rat and SD rat,and the relative expression of NF-?B1 and MAPK14 mRNA in spleen of plateau zokor was significantly lower than that of SD rat.The above results showed that plateau zokor has a smaller spleen than the other two rodent species to reduce the innate immunity investment in the spleen,because the plateau zokor lives in oxygen-deficient tunnels and has a lower antigenic risk.The plateau zokor maintains immune homeostasis to adapt to the environment in tunnels,resulting in the suppression of the LPS-mediated TLR4 signaling pathway.We also noted high levels of HIF-1?in the spleens of plateau zokors,suppressing its crosstalk with TLR4 in promoting the innate immune response.Three species of peritoneal macrophages were isolated and perform CoCl2simulated hypoxia culture after LPS immune stress,the results showed that the phagocytic activity of macrophages of plateau zokor was lower than that of bamboo rat and SD rat,especially in hypoxia treatment.The level of ROS in macrophages of plateau zokor was significantly lower than that of the SD rat after hypoxia treatment.Hypoxia treatment after LPS immune stress,the phagocytic activity of macrophages of the three species decreased.The plateau zokor had the largest decrease and was at the lowest level,and the level of ROS production was at the lowest level.When three species of macrophages were exposed to hypoxia,the apoptotic rate of macrophages of zokor was significantly lower than that of bamboo rat and SD rat,the expression level of pro-apoptotic gene Bax was lower than that of bamboo rat and SD rat,the expression level of anti-apoptotic gene Bcl-2 was higher than that of bamboo rat and SD rat,and the ratio of Bcl-2/Bax was higher than that of bamboo rat and SD rat,which makes its susceptibility to apoptosis was weaker than that of bamboo rat and SD rat.The key molecule Caspase-3 activity was also significantly lower than that of bamboo rat and SD rat.The apoptosis of macrophages of three species increases after LPS immune stress,especially in plateau zokor.LPS combined with hypoxia aggravated the apoptosis levels of three species of macrophages,but the plateau zokor was still significantly lower than that of bamboo rat and SD rat.The level of HIF-1?in macrophages of plateau zokor was significantly higher than that of bamboo rat and SD rat.Additionally,the amount of LDH,IL-1?and IL-18 released into the supernatant from macrophages of plateau zokor was lower than that of bamboo or SD rat,and the expression level of Gasdermin D in macrophages was lower than that of the other two species.The activity of Caspase-1 and Caspase-4 in macrophages of plateau zokor was significantly lower than that of bamboo rat and SD rat.LPS stimulated macrophages,resulting in a strong pro-inflammatory response of macrophages,which can be demonstrated by the significant increase of Caspase-1 and Caspase-4 activities compared with the saline control group.LPS combined with hypoxia increased the level of macrophage cell pyroptosis.However,the macrophages of plateau zokor showed a weaker pyroptosis level than that of bamboo rat and SD rat.The above results showed that hypoxia may weaken the function of macrophages of plateau zokor compared with that of bamboo rat and SD rat as a whole,resulting in heterogeneity.The phagocytic activity,apoptosis and pyroptosis level of macrophages of plateau zokor are weaker than those of bamboo rat and SD rat,and LPS combined with hypoxia aggravates the development of this trend.To conclude,our present study explored in detail the innate immune function of plateau zokor and the innate immune heterogeneity with the other two species and its mechanism.The results will help to elucidate the immune adaptation mechanism of high-altitude underground rodents,provide basic data for further study of immune function of high-altitude underground rodents,and the interaction between extreme environment and animal immune function,and also provide a reference for high altitude medicine. |
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