| Malaria which caused by Plasmodium app. has been one of the six devastating tropical parasite infectious diseases popular around the world and one of the top five parasitic disease in China. Malaria is an acute febrile disease, the symptoms such as fever, headache, chills and vomiting may be milder and therefore difficult to detect at the very beginning. However, immunocompromised people such as children and pregnant women are susceptible to severe malarial syndromes such as severe anemia,acute respiratory distress, and clinical jaundice, with the brain, lungs, kidney, and liver all variously affected and finally lead to death. Since population flow,environmental change and anti-malaria drug resistance, the malaria threaten to human health still cannot be ignored.Varying degrees of liver dysfunction can be generally observed during malaria infection, while severe malaria can lead to the failure and necrosis of liver, which is closely related to the infiltration and pathological immune response of inflammatory cells in the liver. Kupffer cells(KCs) are specialized macrophages located in the liver that lining the walls of the sinusoids. On one hand KCs constitute a defense barrier in the liver, on the other hand the recruitment of pro-inflammatory cytokines secreted by immune cells contribute to the liver injury. Since blood stage is important for malarialinfection, Plasmodium invasion, proliferation and lysis of red blood cells by the liver surface specific markers of microvascular during this time, which prompted a large number of KCs aggregation. This process often leads to liver injury, which indicated that the infiltration and proliferation of monocyte-macrophage cells participate in immune pathological reactions in the liver. However, the invasion of Plasmodium lead to the transcription of type one IFNs(IFN-?/?) gene in hepatocytes, which stimulate recruitment of leukocytes into the liver by expressing IFN-stimulated genes(ISGs).This indicate holding the homeostasis between various types of inflammatory cytokines is very important to the against of malarial infection. But the specific pathological immune mechanisms of KCs in the liver injury during malarial infection still remain uncleared.Tim-3 is a member of immunoglobulin mucin molecules family which mainly express on Th1 cells, maintain an inhibition of Th1 cells mediated immune response.Galectin-9(Gal-9), a member of lectin family, is the main natural ligand of Tim-3which mainly express on CD4+ T cell, can selectively binds with ?-galactosidase and interact with Tim-3 which can induce the apoptosis of Th1 cell and peripheral immune tolerance. The interaction between Tim-3 and Gal-9 can promote macrophage phagocytosis of pathogens and inhibit T cell responses, which lead to the apoptosis of T cells gradually during acute inflammations and rebuild the function of CD8+ T cells during chronic inflammation, showing the important role of Tim-3/Gal-9in inflammations. It has been reported that the up-regulation of Tim-3 on the NKT cells induced the expression of Gal-9 on KCs, while KCs secret IFN-? leads to the apoptosis of Tim-3+ NKT cells, which limit the inflammatory response and avoid destructive immunity.However, the role of Tim-3 and Gal-9 in the liver injury caused by Plasmodium is still uncleared. Our study focuses on the Tim-3/Gal-9 signal pathway on KCs that involves in the liver injury of a murine malarial model, which may provide a new therapeutic target for liver injury of malaria infection.Purpose and significanceIn this paper, mice were infected with Plasmodium berghei ANKA(Pb ANKA)to research the immune regulation role on macrophages in the liver by blockade of galectins. Our results show that Tim-3/Gal-9 pathway on KCs may be involved in the liver immune pathological regulation of Pb ANKA.Part one1) Content and methods(1) The establishment of a murine malarial modelKunming(KM) mice were infected with 106 Pb ANKA.(2) The packet and observation of malarial micePb ANKA-infected mice were sacrificed by CO2 asphyxiation for obtain the livers and spleens at days 5, 10, 15, and 20 post infection(p.i.). Survival statuses were observed and parasitemia counts were performed with a hematocytometer on the Giemsa-stained thin blood smears.(3) The measurement of parasite burdens for malarial miceParasite burdens were detected through measuring the m RNA level of Pb ANKA18 S r RNA by using quantitative real-time PCR(q RT-PCR) in different time points after infection.(4) The inflammatory histopathological analysis of malarial miceSections of liver and spleen tissues in different time points after infection were stained with hematoxylin and eosin(H&E) and the pathological change of liver injury was assessed by using semi-quantitative histopathological analysis.(5) The measurement of Tim-3 and Gal-9 positive cells for malarial miceImmunohistochemistry was used for detecting the expression of Tim-3 and Gal-9in the livers and spleens, and semi-quantitative analysis were used for counting the positive staining cells.(6) The measurement of cytokines for malarial miceTim-3, Gal-9, and inflammatory cytokines(IL-1?, IL-6, TNF-?, IL-10, and IL-4)m RNA expression were measured by using q RT-PCR in different time points after infection.2) Result(1) KM mice died between day 7 and 20 post infection(p.i.) and the parasitemia reached 62.61 to 78.63 % when deaths occurred.(2) The parasite burdens in the livers and spleens were significantly increased at all the time point after infection. Compared with day 5 p.i., the parasite burden in livers and spleens were significantly increased at days 10(P<0.05), 15(P<0.001), and20(P<0.001) p.i.; compared with day 10 p.i., the parasite burden in livers and spleens were significantly increased at days 15(P<0.001) and 20(P<0.001 and P<0.05,respectively) p.i.(3) Varying degrees of pathological damage were occurred in the liver and spleen tissues of infected mice at all time points. The histopathology scores in the livers and spleens were significantly increased at all the time point after infection.(4) The positive cells of Tim-3 and Gal-9 in the livers and spleens were significantly increased at all the time point after infection.(5) Compared with uninfected controls, Tim-3 m RNA levels in the livers of the malarial mice were significantly increased at all the time point and the Tim-3 m RNA levels in the livers at day 20 p.i. were significantly higher than other time point(P<0.001). Compared with uninfected controls, Gal-9 m RNA levels in the livers of the malarial mice were significantly increased at days 5 and 10 p.i.(P<0.001).Compared with uninfected controls, Gal-9 m RNA levels in the spleens were significantly increased at days 10 and 15 p.i.(P<0.001 and P<0.05, respectively).(6) Compared with uninfected controls, the inflammatory cytokines m RNA levels of IL-1?, IL-6, TNF-?, and IL-10 were significantly increased while the m RNA levels of IL-4 were significantly decreased after infection in the livers. Compared with uninfected controls, the inflammatory cytokines m RNA levels of IL-6 and IL-10 were significantly increased while the m RNA levels of IL-1? were significantly decreased after infection in the spleens.3) ConclusionAfter infected with Pb ANKA-i RBCs, with the extension of time, the parasite burdens and histopathological changes in the livers and spleens of the mice were significantly increased, while the positive cells of Tim-3 and Gal-9 in the livers and spleens were also increased, which suggesting the Tim-3/Gal-9 signal pathway may be involved in the pathological and immune regulation of liver injury during malarial infection.Part two1) Content and methods(1) The establishment of a murine malarial modelKM mice were infected with 106 Pb ANKA.(2) The packet and observation of malarial mice treated with or without?-lactoseMalarial and uninfected mice treated with or without 300 m M of ?-lactose every12 hours. Mice were sacrificed by CO2 asphyxiation for obtain the livers at days 5 and7 p.i. Survival statuses were observed and parasitemia counts were performed with a hematocytometer on the Giemsa-stained thin blood smears.(3) The measurement of parasite burdens for malarial mice treated with or without ?-lactoseParasite burdens were detected through measuring the m RNA level of Pb ANKA18 S r RNA by q RT-PCR at days 5 and 7 p.i.(4) The inflammatory histopathological analysis of malarial mice treated with or without ?-lactoseSections of liver tissues were stained with H&E. The pathological change of liver injury was assessed by using semi-quantitative histopathological analysis.(5) The measurement of KCs positive cells for malarial mice treated with or without ?-lactoseSections of liver tissues were stained with immunohistochemistry of CD68 andthe semi-quantitative analyses were used for the positive staining cells.(6) The immunofluorescence double-staining of CD68 with Tim-3 and CD68 with Gal-9 for malarial mice treated with or without ?-lactoseSections of liver tissues were stained with immunofluorescence double-staining of CD68 with Tim-3 and CD68 with Gal-9 on liver tissues were performed.(7) The measurement of murine macrophage RAW264.7 cells co-cultured with Pb ANKA-i RBCsMurine macrophage RAW264.7 cells was cultured 24 h with Pb ANKA-i RBCs at parasite to host ratios(multiplicity of infection, MOI) 10:1, then the m RNA expression of Tim-3, Gal-9, IFN-? and IFN-? were measured by q RT-PCR. After blockade galectins with ?-lactose, the m RNA expression of IFN-? and IFN-? were measured by q RT-PCR.(8) The measurement of type one and two IFNs for malarial mice treated with or without ?-lactoseType one(IFN-?/?) and two(IFN-?) IFNs were detected by q RT-PCR at days 5and 7 p.i.(9) The measurement of cytokines for malarial mice treated with or without?-lactoseInflammatory cytokines(IL-6, IL-12, TNF-?, and IL-10) were detected by q RT-PCR at days 5 and 7 p.i.(10) The apoptosis assay in the livers for malarial mice treated with or without ?-lactoseApoptosis cells were measured by TUNEL staining and accessed by semi-quantitative analysis.2) Result(1) The malarial mice which suffered from severe malaria were died between days 7 and 10 p.i. while the malarial mice treated with ?-lactose died between days 5and 7 p.i. Parasitaemia in the malarial mice treated with ?-lactose was significantly higher than that of the malarial ones at day 4, 5 and 6 p.i.(P<0.05).(2) The parasite burdens in the livers of malarial mice treated with ?-lactose were higher than the malarial ones both at day 5 and 7 p.i.(3) The pathological damage and histological scores in the livers of malarial mice treated with ?-lactose were higher than the malarial ones both at day 5 and 7 p.i.(4) Compared with uninfected mice treated with or without ?-lactose, the numbers of positive KCs in liver tissues of the malarial mice treated with or without?-lactose were significantly increased both at 5 and 7 days p.i., while the numbers of posistive KCs in liver tissues of the malarial mice treated with ?-lactose were higher than the malarial ones both at 5 and 7 days p.i.(P<0.001).(5) Both Tim-3 and Gal-9 were expressed on KCs in the livers of each group.Compared with uninfected mice treated with or without ?-lactose, much more and stonger Tim-3+ CD68 and Gal-9+ CD68 cells were observed in the malarial mice treated with or without ?-lactose, while the Tim-3+ CD68 and Gal-9+ CD68 cells were much more stonger in the malarial mice treated with ?-lactose than the malarial ones.(6) Compared with murine macrophage RAW264.7 cells controls, the Tim-3/Gal-9 m RNA levels of the cells co-cultured with Pb ANKA-i RBCs 24 h were significantly increased(P<0.001). Compared with murine macrophage RAW264.7cells co-cultured with or without ?-lactose, the IFN-?/? m RNA levels of the cells co-cultured with Pb ANKA-i RBCs 24 h were significantly increased(P<0.001), while the IFN-?/? m RNA levels of the cells co-cultured with Pb ANKA-i RBCs treated with?-lactose 24 h were lower than the ones co-cultured treated without ?-lactose(P<0.001).(7) Compared with uninfected mice treated with or without ?-lactose, the IFN-?/? m RNA levels in the livers of the malarial mice treated with or without?-lactose were significantly increased at day 5 p.i.(P<0.001) and the malarial mice treated with ?-lactose were lower than the malarial ones(P<0.001), while IFN-?/?m RNA levels in the livers of the malarial mice treated with ?-lactose were also lower than the malarial ones at day 7 p.i.(P<0.05). Compared with uninfected mice treated with or without ?-lactose, the IFN-? m RNA levels in the livers of malarial mice treated with or without ?-lactose were significantly increased at days 5 and 7 p.i., andthe malarial mice treated with ?-lactose were lower than the malarial ones at day 7 p.i.(P<0.05).(8) Compared with uninfected mice treated with or without ?-lactose, the inflammatory cytokines m RNA levels of IL-6, IL-12, TNF-?, and IL-10 were significantly increased in the livers of the malarial mice trated with or without?-lactose at days 5 and 7 p.i., while the malarial mice treated with ?-lactose were higher than the malarial ones.(9) Compared with uninfected mice treated with or without ?-lactose, apoptotic cells were frequently observed both in the livers of malarial mice treated with or without ?-lactose at days 5 and 7 p.i.(P<0.001). Much more apoptotic cells in the livers of malarial mice treated with ?-lactose than the malarial ones at days 5 and 7 p.i.were also observed(P<0.001).3) ConclusionCompared with malarial mice, CD68+ KCs in the livers of the malarial mice treated with ?-lactose were significantly increased while Tim-3/Gal-9 expressions on the KCs were also significantly enhanced, accompanied by a decline expressions of IFN-?/?, inflammation and apoptosis was significantly increased in the liver. Our study suggest that Tim-3/Gal-9 may be involved in the immune regulation of raising KCs and secrete IFN-?/? to change the secretion of inflammatory cytokines and apoptosis in the liver and eventually take a part in the immune regulation during malaria induced liver injury. |
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