The Roles Of Galectins In Acute Lung Injury Induced By Plasmodium Berghei ANKA In A Mouse Model

BackgroundMalaria is still a major global health problem. Plasmodium infection may result in severe malaria in patients, which can develop malaria-associated ALI/ARDS and often results in high mortality. Although the participation of lungs involved in the severity of malaria has been well documented, knowledge about this pathogenesis is still limited. ALI develops before the onset of cerebral malaria symptoms, and malaria-induced ALI pathophysiology demonstrates that inflammatory-mediated increased capillary permeability or endothelial damage leads to diffuse alveolar destruction that can continue after parasite clearance[1]. However, few studies have addressed the characteristics of the immunological response during malaria-induced ALI.Galectins are a family of highly-conserved glycan-binding proteins that play an important role in the innate and adaptive immune responses. Previous works have reported that the major galectins expressed in the lung are Gal-1,-3,-8, and-9[2-5], and galectin-9/Tim-3 pathway plays an important role in virus, bacterium and parasite infection. The galectin-9/Tim-3 interaction acts as a specific inhibitor of Th1 and Th17 immune responses[6]. Besides, galectin-9 can also bind to other receptors including CD44, CD137, and PDI[7-9]. However, the roles of galectins in malaria-induced ALI are largely unknown.Purpose and significanceThis study sought to explore the roles of galectins in acute lung injury induced by Pb ANKA in a mouse model. Our data demonstrated galectin-9 and its receptors as important factors in the development of malaria-induced ALI, and provided important evidence for the prevention and treat of it.Part IMethods and Materials:Mice and experimental infections Female Kunming mice were injected i.p. with 106 Pb ANKA-infected red blood cells(i RBCs) to establish mouse model of malaria-induced ALI.Parasitemia Parasitaemia developments in Pb ANKA-infected mice were monitored daily by microscopic examination of Giemsa-stained thin blood smears of tail blood.Histopathology Pb ANKA-infected mice were sacrificed by CO2 asphyxiation for examination.Lungs and MLNs from Pb ANKA-infected mice were stained with HE, and evaluated for histological changes.Immunohistochemical staining for galectin-9 and Tim-3 in the mouse lungs and MLNs Immunohistochemistry was carried out using the streptavidin–biotin–peroxidase complex(SABC) method. Sections were incubated with rabbit anti-Tim-3, and anti-galectin-9. Tim-3- and galectin-9-positive cells were identified by dark-brown staining.Measurement of m RNA expression using quantitative real-time PCR(q RT-PCR)Total RNA was extracted from mouse lung and MLN tissue. To determine tissue m RNA levels of galectin-9, Tim-3, IL-6, IL-10, and TNF-?,q RT-PCR was performed.Results Malaria-associated acute lung injury(ALI)Pb ANKAinfected mice died between 8 and 22 days p.i. Approximately 32% of Pb ANKA-infected mice succumbed between 8 and 10 days p.i.developing severe malaria with neurological signs(cerebral malaria, CM); the remaining mice died between 18 and 22 days p.i. with hyper-parasitemia and Dyspnea(non-CM). The total protein levels in the BALFs of mice with non-CM were significantly elevated when death occured, indicating that a disruption of the alveolar-capillary membrane barrier and ALI occurred as a result of Pb ANKA infection.Lung and MLN histopathology The lung tissues of mice were examined histologically. Moderate to severe inflammatory cellular infiltration(neutrophil-dominant and foamy macrophages in the alveolar and interstitial sites), alveolar edema, hemorrhage, thickening of the alveolar septum, and highly parasitized RBCs were noted in the lungs of both mice infected with Pb ANKA at days 5, 10, 15, and 20 p.i. The loss of normal architecture(cortical and medullary regions) and parasitized RBCs were noted in the MLN sections of mice infected with Pb ANKA at days 5, 10, 15, and 20 p.i.Immunohistochemical staining for galectin-9 and Tim-3 in the mouse lungs and MLNs There was few galectin-9+ and Tim-3+ cells in the lung tissues and MLN sections of uninfected controls. However, in mice infected with Pb ANKA, there were marked increased galectin-9+ and Tim-3+ cells at days 5, 10, 15, and 20 p.i. in both lung tissues and MLN tissues.galectin-9 and Tim-3 m RNA expressions in the lungs and MLNs Compared with uninfected controls, galectin-9 m RNA expressions were significantly up-regulated at days 5(P< 0.001), 10(P< 0.01), 5(P< 0.05) and 20(P<0.001) p.i. in the lungs and at days 15(P< 0.05) and 20(P< 0.001) p.i. in the MLNs of Pb ANKA-infected mice; Tim-3 expressions in the lungs and MLNs ofPb ANKA-infected mice were significantly up-regulated at days 10(P< 0.05), 15(P<0.01), and 20(P< 0.001 and P< 0.01, respectively) p.i.Determination of pro- and anti-inflammatory cytokine responses in lungs and MLNs Compared with uninfected controls, the expressions of TNF-? were significantly increased in lungs at day 15(P< 0.01) but significantly decreased in MLNs at days 5(P< 0.05), 10(P < 0.01), and 20(P< 0.01) p.i.; IL-6 m RNA expressions were significantly increased in lungs at days 10(P< 0.001), 15(P < 0.05), and 20(P< 0.05)p.i. and in MLNs at days 5 and 10 p.i.(P< 0.001); IL-10 expressions were significantly increased in lungs and MLNs at days 10(P< 0.01), 15(P< 0.001 and P<0.05, respectively), and 20(P< 0.01 and P< 0.05, respectively) p.i.Conclusions:We studied galectin-9 and Tim-3 expression, immunological, and pathological relevance in a mouse model. Galectin-9/Tim-3 may play an important role in the development of ALI in Pb ANKA-infected mouse model.Part IIMethods and Materials:Mice and experimental infections Female Kunming mice were injected i.p. with 106 Pb ANKA-infected red blood cells(i RBCs) to establish mouse model of malaria-induced ALI. Some mice were injected intraperitoneally(i.p.) with 100?l 300 m M of ?-lactose solution in PBS twice daily starting from day 1 post infection(p.i.) until the day mice were sacrificed.Mortality was monitored daily.Parasitemia Parasitaemia developments in Pb ANKA-infected mice were monitored daily by microscopic examination of Giemsa-stained thin blood smears of tail blood.Histopathology Pb ANKA-infected mice were sacrificed by CO2 asphyxiation for examination.Lungs from Pb ANKA-infected mice were stained with HE, and evaluated for histological changes.Immunohistochemical staining for CD68 in the mouse lungs and MLNs Immunohistochemistry was carried out using the streptavidin–biotin–peroxidase complex(SABC) method. Sections were incubated with rabbit anti-CD68.CD68-positive cells were identified by dark-brown staining.Measurement of m RNA expression using quantitative real-time PCR(q RT-PCR)Total RNA was extracted from mouse lung and MLN tissue. To determine tissue m RNA levels of galectin-1, 3, 8, and 9, the receptors of galectin-9(Tim-3, CD44,CD137 and PDI), HMGB1(a ligand for Tim-3) and HAS1-3(HA, a ligand for CD44),IL-4, IL-10, and IFN-?,-?, and-?, q RT-PCR was performed.Immunofluorescence assay(IFA) of BALF cells and lung tissues BALFs were obtained by instillation and aspiration of 0.6 ml aliquots of PBS from Pb ANKA-infected mice and then spun at 800 g at 4 ? for 5 min, and the pelleted cells were resuspended and seeded on glass coverslips in 24-well plates. The cells and lung tissue sections were incubated with mouse anti-mouse CD68 and rabbit anti-Tim-3 or rabbit anti-galectin-9. CD68+macrophages were identified by their green fluorescent, whereas Tim-3 and galectin-9 appeared as red fluorescent located in the nucleus or cytoplasm, once CD68 and galectin-9 or CD68 and Tim-3superimposed in one image that would appear as yellow fluorescent.Apoptosis assay Signals of apoptosis in 5-?m lung paraffin sections were detected using TUNEL assay. TUNEL-positive cells were determined under high power field as well as the area of each field(0.015066 mm2).Isolation and culture of peritoneal macrophages Peritoneal macrophages were isolated from KM mice and were co-cultured with5×106 Pb ANKA-i RBCs for 24 h. To block the binding of autocrine Gal-9 and its receptors, cells were pre-incubated with 30 m M ?-lactose for 1 h before adding Pb ANKA-i RBCs. Samples were stored at –80? until subjected to further analysis.Results?-lactose treatment increased BALF total protein, parasitimia, and host mortality rate in mice with CM Infected mice treated with ?-lactose had 100% mortality by day 7 p.i. compared with untreated infected mice, in the Pb ANKA-infected mice treated with ?-lactose,the total protein levels in the BALFs were significantly elevated at both days 5 and 7p.i.(P < 0.01),the parasitaemia was also significantly higher at days 4, 5, and 6 p.i. but not at day 7(P > 0.05),and there was significantly increased lung parasite burden at day 5(P < 0.05) but not at day 7(P > 0.05) p.i.?-lactose treatment promotes lung pathology in Pb ANKA-infected mice Compared to the untreated untreated malarial mice, increased cell infiltration including lymphocytes, mononuclear cells, and neutrophils was observed in lungs of malarial mice treated with ?-lactose at day 5 p.i., leading to severe inflammation and tissue damage at day 7 p.i. in the ?-lactose treated malarial mice.m RNA expressions of galectins, the receptors of galectin-9 and their ligands, and IFN-I, IFN-II, IL-4, IL-10.Previous works have reported that the major galectins expressed in the lung are galectin-1,-3,-8, and-9. We next measured m RNA levels of galectin-1,-3,-8, and-9and found that only galectin-9 was significantly increased in the lungs of both infected mice and infected mice treated with ?-lactose at days 5 and 7 p.i.(P < 0.01),while galectin-3,-8, and-9 were significantly increased in the MLNs.The receptors of Gal-9 including Tim-3, CD44, CD137, and PDI were measured in the present study.All of them were significantly increased in the lungs of both infected mice and infected mice treated with ?-lactose at days 5 or 7 p.i. Interestingly,after ?-lactose treatment, HMGB1(a ligand for Tim-3) and HAS1-3(HA, a ligand for CD44) were significantly increased in both the lungs and MLNs.Compared with malarial groups, significantly increased levels of IFN-?,-?, and-?in lungs of malarial mice treated with ?-lactose were observed at day 5 p.i.(P <0.001); however, there were significantly decreased expressions of IFN-?(P < 0.05)and-?(P < 0.001) in the lungs of ?-lactose treated mice at day 7 p.i. In the MLN tissues, there were significantly decreased expressions of IFN-?(P < 0.05) and-?(P <0.001) at day 5 p.i., and significantly decreased expressions of IFN-?(P < 0.01),-?(P< 0.001), and-?(P < 0.001) at day 7 p.i. in malarial mice treated with ?-lactose.Compared with malarial group, there were significantly increased levels of IL-4 in the lungs at day 5 p.i.(P < 0.05), but significantly decreased levels of IL-4 in the MLNs at day 7 p.i.(P < 0.05), and significantly increased levels of IL-10 in both lungs(P <0.05) and CLNs(P < 0.001) at day 5 and in the lungs at day 7 p.i.(P < 0.05) of malarial mice treated with ?-lactose.Treatment with ?-lactose increased CD68+ macrophages in the lung of Pb ANKA-infected mice Compared with uninfected controls, there were significantly higher numbers of CD68+ macrophages in the lungs of Pb ANKA-infected mice and significantly more CD68+ macrophages in the lung tissues of malarial mice treated with ?-lactose than those of untreated malarial mice at day 7 p.i.Galectin-9 and Tim-3 expressions on ex-vivo macrophages from BALFs and in lung tissues The results showed that both galectin-9 and its receptor Tim-3 were expressed on macrophages in this model. Compared with controls(uninfected and untreated/uninfected and treated), stronger galectin-9 and Tim-3 signals were observed on BALF CD68+ macrophages from both malarial mice and malarial mice treated with ?-lactose. Similarly, both galectin-9 and Tim-3 were expressed on the CD68+ macrophages in the lung tissues of all the groups.Blockage of galectins induces apoptosis in the lungs during ALI The numbers of apoptotic cells in malarial mice treated with ?-lactose were significantly higher than those in malarial mice at days 5(P < 0.01) and 7(P < 0.001)p.i. Malarial mice treated with ?-lactose had more apoptotic cells in lungs at day 7 p.i.than those at day 5 p.i.(P < 0.05).Blockade of galectin-9/Tim-3 pathway led to increased IFN-I production inperitoneal macrophages co-cultured with Pb ANKA-i RBCs in vitro Compared with the control cell group, the m RNA expressions of galectin-9, Tim-3,HAS1, HAS2, HAS3, IFN-I(IFN-?, ?), IFN-II(IFN-?), and IL10 were all significantly increased in the peritoneal macrophages co-cultured with Pb ANKA-i RBCs(P < 0.05).The m RNA levels of IFN-? and-? were further significantly increased(P < 0.05), and the level of HMGB1 was also significantly increased(P < 0.05) in the macrophages co-cultured with Pb ANKA-i RBCs after?-lactose treatment.Conclusions:The outcome of the current study demonstrates that blockade of galectins by?-lactose, especially the galectin-9(Tim-3, CD44, CD137, and PDI) signaling affected increased parasitemia/tissure parasite burden, exacerbated lung damage, and shortened the survival of ?-lactose-treated malarial mice as compared to malarial mice during ALI, likely mediated by the elevated expressions of IFN-? and-?, and IL-10 through galectin-9 independent and Tim-3-HMGB1 interaction in the macrophages induced by Pb ANKA. Our data elucidate a new mechanism by which the Tim-3-HMGB1 interaction regulates macrophage activation after blockade of galectins. In addition, it indicated the importance of galectin-9 and its receptors signaling during malaria-induced ALI caused by Pb ANKA.