• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

    2021-12-17 11:53:14YaoLiYueZhaoXuelingXuRuiZhangJinlongZhangXiaoshengZhangYanLiShoulongDengandZhengxingLian

    Yao Li,Yue Zhao,Xueling Xu,Rui Zhang,Jinlong Zhang,Xiaosheng Zhang,Yan Li,3*,Shoulong Dengand Zhengxing Lian*

    Abstract

    Background:Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health.Toll-like receptor 4(TLR4)plays a crucial role in host defence against Gram-negative bacteria.Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections.In a previous study,we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance.The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood.

    Results:In the present study,we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model.The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection.This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis.Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4,Src signalling,or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria,weakened the ability of the monocytes to eliminate the bacteria,and increased the pH of the endosomes.

    Conclusion:Together,our results reveal the effects of TLR4 on the control of E.coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model,providing theoretical support for breeding disease resistance in the future.Furthermore,Src and caveolin 1(CAV1)could be potentially valuable targets for the control of infectious diseases.

    Keywords:Caveolae-dependent endocytosis,Host defence infection,Inflammatory responses,Monocytes,Toll-like receptor 4

    Background

    Toll-like receptor 4(TLR4)is known to be a patternrecognition receptor that triggers innate immunity[1–3].This molecule is expressed mainly in the immunocytes of mammals,including macrophages,monocytes,neutrophils,and dendritic cells[4].As an important component of the body’s defence system,monocytes not only promote specific proinflammatory and anti-inflammatory processes but also endocytose and eliminate pathogens to maintain cell homeostasis[5].TLR4 recognizes Gram-negative bacteria via their signature molecules to activate the host’s innate immune response,which is tightly regulated via distinct factors and pathways.The localization of TLR4 has emerged as a key determinant of TLR4 function.These factors include subcellular TLR4 localization and ligand sensing,which occurs on the cell surface through activation of the Toll-interleukin 1 receptor(TIR)domain containing adaptor protein(TIRAP)-myeloid differentiation factor 88(MyD88)-dependent pathway and through endocytosis that activates the TRIF-related adaptor molecule(TRAM)-TIR domain containing adaptor-inducing interferon-β(TRIF)-dependent pathway[6].TLR4 does not simultaneously activate the MyD88-and TRIF-dependent pathways;instead,it induces these two signalling pathways sequentially following the endocytosis of pathogens[7].First,when bacteria bind to TLR4-bound myeloid differentiation protein 2(MD2)on the plasma membrane,TIRAP and MyD88 participate in the rapid activation of the TIRAP-MyD88-dependent pathway,which results in the production of proinflammatory cytokines[8].Next,TLR4 enters the endocytosis pathway by being translocated away from the cell surface to the endosome accompanied by cargo[9].With the help of the bridging factor TRAM,TLR4 recruits the TRIF receptor that drives the TRAM-TRIF-dependent pathway and leads to the production of type I interferons[10].

    The endocytosis of pathogens has emerged as a critical control step in the antimicrobial signal transduction process[11].Macrophages with defects in the internalization of cell surface TLR4 exhibit enhanced inflammatory cytokine production following stimulation with lipopolysaccharide(LPS)and fail to induce LPS tolerance despite repeated LPS stimulation[12].The internalization of TLR4 into the endosome is one of the negative regulatory mechanisms that prevent an excessive inflammatory response[13].

    After immunocytes internalize invading pathogens by endocytosis,a series of vesicular trafficking steps occur with organelles ranging from early endosomes to lysosomes.At the end of this process,the cargo can be effectively degraded when mature endosomes fuse with endolysosomes[14].Acidification is essential during endosome maturation:a sufficiently low pH is a prerequisite for endosome-lysosome fusion and provides optimal conditions for the activity of certain hydrolytic enzymes[15].TLR4 is reported to control the rhythm of endosomal maturation and to guide bacteria into the “fast lane”of antigen presentation compared to apoptotic cell(not involved in TLR4)degradation by phagocytes.The inhibition of TLR4 downstream signalling events,such as mitogen-activated protein kinase(MAPK),blocks this enhanced endosomal maturation[16].Research on Salmonella has found that TLR4 signalling enhances the acidification rate of endosomes containing Salmonella[17].However,the mechanisms linking TLR4 and endosome maturation remain to be fully explored.

    Endocytosis occurs via a variety of mechanisms in mammalian cells,including clathrin-dependent endocytosis,micropinocytosis,phagocytosis,and caveolae-dependent endocytosis[18,19].Caveolae are submicroscopic plasma membrane lipid rafts that are rich in cholesterol and sphingolipids and found in many mammalian cell types[20].Caveolae have vital functions in signal transduction and the elimination of bacteria[21].TLR4 activation has been associated with the translocation of TLR4 and signalling proteins into caveolae since the inhibition of the caveolae-dependent endocytosis pathway blocks LPS-induced TLR4 signalling[22].Caveolin 1(CAV1)is the principal structural and signalling component of caveolae and has been used as a marker protein[23].This molecule has been shown to be involved in endocytosis.The findings from a mouse model of sepsis indicated that CAV1 is an important protective modulator of sepsis,as CAV1 may regulate inflammation and reduce the bacterial burden[24].Studies on mouse macrophages found that knocking out CAV1 reduces their capacity for endocytosis and their ability to kill bacteria.In addition,the expression of TLR4 and MyD88 was decreased,and the production of inflammatory cytokines was also decreased[25].Several studies have found that CAV1 can be phosphorylated by the tyrosine kinase Src,and activated CAV1 promotes caveolae-mediated endocytosis[21,26].Furthermore,the Src kinase family phosphorylates TLR4 to dissociate MyD88 and MyD88 adapter-like(MAL)/TIRAP,thereby suppressing LPS-induced inflammatory responses[27].These results indicate the potential role of TLR4 in caveolae-dependent endocytosis and signalling,although the precise mechanisms remain unknown.

    In our previous study,we bred transgenic sheep overexpressing TLR4[28,29].These transgenic sheep produced a stronger inflammatory response during the early stages of bacterial infection than control sheep,and the infection subsided quickly.TLR4 is known to increase the expression of scavenger receptors,which enhance the adhesion capacity of immunocytes and contribute to the endocytosis of pathogenic bacteria[30,31].However,the effect of TLR4 overexpression on immune defence mechanisms has not been completely elucidated.Here,we continued this line of investigation by determining the effects of TLR4 on bacterial endocytosis and the bactericidal ability.We further examined whether the immunocytes of transgenic sheep have improved antibacterial ability via caveolae-dependent endocytosis.

    Methods

    Sheep

    The founder transgenic sheep were produced through microinjection of a linear vector containing sheep TLR4(Fig.1a)into the pronucleus of fertilized eggs[28,29].Founder transgenic sheep were bred with wild-type sheep.Among the offspring of the transgenic sheep,healthy male 2-to 3-year-old sheep were identified as either wild-type or transgenic by Southern blotting.Genomic DNA(20μg)was extracted from the ear tissue and digested with HindIII(New England Biolabs,Ipswich,Britain).The probe used for Southern blotting was generated by PCR with the following primer pair:forward(F),5′-ACTGGTAAAG AACTTGGAGGAGG-3′and reverse(R),5′-CCTTCA CAGCATTCAACAGACC-3′,and the 671-bp PCR product was labelled with digoxigenin(Roche Diagnostics,Mannheim,Germany).Four sheep were included in the transgenic and four sheep in wild-type groups for the subsequent experiments.

    Fig.1 Detection of TLR4 overexpression in ovine blood monocytes.a Schematic diagram of the structure of the CMV-Ovis aries TLR4 overexpression vector.b Representative Southern blot used to identify transgenic(TG)sheep based on the presence of the TLR4 transgene.Partially TG sheep had both the endogenous 4700-bp TLR4 band and the exogenous 2771-bp TLR4 band.Marker,1-kb ladder;lanes 1–8,eight individuals comprising the wild-type(WT)sheep in lanes 1 and 3 and the TG sheep in lanes 2 and 4–8.c Monocytes were isolated from four WT and four TG sheep and stained for the expression of the monocyte markers,CD14 and CD11b.DAPI was used to stain the cell nuclei.Scale bar:100μm.d Similarly,TLR4 expression in the TG and WT monocytes was examined by immunofluorescence microscopy.Scale bar:100μm.e The protein expression of TLR4 in monocytes was examined by western blotting.f Quantification of the data in(e).g The mRNA expression of TLR4 in monocytes was evaluated by qRT-PCR.Both the mRNA and protein expression levels of TLR4 were significantly higher in the TG monocytes than in the WT monocytes.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.GAPDH was used for normalization

    Cells and culture conditions

    The initial peripheral blood mononuclear cells were isolated from the peripheral blood of sheep that had been collected aseptically from the jugular vein in separation medium(TBDscience,Tianjing,China),followed by density gradient centrifugation(2,000×g for 30 min).The cells were seeded at a density of 1×106cells per well in 6-well culture plates.After incubation at 37°C in a 5% CO2incubator for 2 h,the nonadherent cells were removed by washing the wells three times with phosphatebuffered saline(PBS).The adherent cells were cultured at 37°C in a 5% CO2incubator with RPMI-1640 medium(Gibco,Grand Island,NY,USA)containing 10% foetal bovine serum(FBS;Gibco).After a further 48h of incubation and the removal of nonadherent cells,the adherent cells were mainly composed of monocytes.

    Immunofluorescence assay

    First,the monocytes were washed with PBS and then digested with 0.25% trypsin-EDTA(Gibco,Grand Island,NY,USA)at 37 °C for 5–10 min.After termination of the digestion,the cell suspension was poured into a 15 mL centrifuge tube and washed twice with PBS,RPMI-1640 medium was added,and a cell counting plate was used for counting.Monocytes were seeded on 8-well glass chamber slides(Millipore,Massachusetts,USA)at a density of 1×105cells per well.After 12h,the cells were washed three times with PBS before being fixed with 4% paraformaldehyde for 20min,followed by blocking with Immunol Staining Blocking Buffer(Beyotime,Beijing,China)for 1h.Diluted primary antibodies against TLR4(1:500;Affinity Biosciences,OH,USA)and cluster of differentiation 14(CD14)and CD11b (1:500;Bioss,Beijing,China)were incubated with the cells at 4°C for 12h and a fluorescein isothiocyanate(FITC)-labelled secondary antibody(1:1,000,Beyotime)was applied according to the manufacturer’s instructions.The cell nuclei were stained with 4′,6-diamidino-2-phenylindole(DAPI;Solarbio,Beijing,China)before placing the coverslip over the slides along with an anti-fluorescence quenching agent.Fluorescent staining was observed with a laser scanning confocal microscope(Nikon Instruments Inc.,NY,USA).

    Western blotting

    The cells were washed three times with PBS before the total protein was harvested with the Minute Total Protein Extraction Kit(Invent Biotechnologies,Eden Prairie,USA).We added phosphatase inhibitor complex I(1:100,Aidlab,China)to lysis buffer(denaturing cell lysis buffer)prior to use.The protein concentration of each extract was quantified using the BCA Protein Assay Kit(Beyotime)according to the manufacturer’s protocols.After the crude lysate had been resuspended in sodium dodecyl sulphate(SDS)buffer and incubated at 95°C for 10min,equal amounts of the protein samples(about20–30μg each)were loaded in separate lanes of a 12% gel for SDS–polyacrylamide gel electrophoresis.Following electrophoretic separation,the proteins were transferred to a polyvinylidene fluoride membrane(Millipore,Massachusetts,USA).The membranes were blocked with 5% milk powder for 2h at room temperature.After incubation overnight with antibodies against TLR4(AF7017,1:500;Affinity Biosciences,OH,USA),Src(2109S,1:1,000;Cell Signaling Technology,Inc.,Boston,USA),p-Src(2101S,1:1,000;Cell Signaling Technology),CAV1(3238S,1:1,000;Cell Signaling Technology),p-CAV1(3251S,1:1,000;Cell Signaling Technology),and GAPDH(D110016,1:5,000;Sangon,Shanghai,China)at 4°C,the membranes were washed thoroughly with Tris-buffered saline containing 0.1% Tween 20(TBST).The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody(A0208,1:5,000;Beyotime).The membranes were visualized with an enhanced chemiluminescence system(Solarbio)according to the manufacturer’s instructions.

    Monocyte infection and colony-forming unit(CFU)counts

    The E.coli K12 strain DH5a and Staphylococcus aureus RN4220 were cultured in Luria-Bertani(LB)broth at 37°C.The bacterial growth phase was measured by the absorbance of the bacterial suspension at 600 nm.An optical density(OD)of~0.4 ensured that bacteria were in the logarithmic growth phase.The number of bacteria was counted by plate counting through serial 10-fold dilutions of the inoculum to LB agar.The bacteria were suspended and diluted to a concentration of 1×107cells/mL in RPMI-1640 medium(Thermo Fisher Scientific,Waltham,USA)containing 10% FBS.Before infection,monocytes were counted again and seeded in 6-well culture plates(Corning,NY,USA).The monocytes were cultured for 12h to ensure cell adherence to the well bottoms,washed three times in PBS without antibiotics,mixed with bacteria at a multiplicity of infection(MOI)of 10,and then centrifuged at 200×g for 2 min.The monocytes were subsequently incubated at 37°C in 5% CO2for the indicated times.The cells were washed three times with PBS containing gentamicin(100 μg/mL)and lysed with 0.1% Triton X-100(Sigma-Aldrich,Saint Louis,USA)to release intracellular bacteria.The total intracellular CFU of bacteria was quantified by culture in agar after serial dilution.

    For determination of the bacterial killing capacity of the monocytes,all samples were first incubated with E.coli for the same period(15 min).The extracellular bacteria were removed by washing the samples three times with PBS containing gentamicin(100 μg/mL).RPMI-1640 supplemented with 10% FBS was added to the cells,and the cells were then incubated for the indicated times.The cells were washed twice with PBS and lysed with 0.1% Triton X-100.The time to phagocytosis was counted from the time of bacterial addition.The data on the numbers of killed bacteria were obtained by subtracting the number of intracellular bacteria at 30 min from the same number at 15min.

    Quantitative reverse-transcription polymerase chain reaction(qRT-PCR)

    The qRT-PCR reactions were performed on an MX3000P instrument(Agilent Technologies,Santa Clara,USA)with the SYBR Premix Ex Taq II kit(TaKaRa,Kyoto,Japan).The total RNA of the sheep peripheral blood monocytes was extracted with an RNA extraction kit(Aidlab,Beijing,China),and a PrimeScript RT kit(TaKaRa)was used to reverse transcribe the RNA into cDNA.Primer pairs were designed by the primer design software Primer 3 and confirmed by the Primer-BLAST tool at the NCBI(National Center of Biotechnology Information)site.A melting curve analysis confirmed the presence of a single gene specific peak and the absence of primer dimers.Melting curve analysis consisted of 95 °C for 15s,60 °C for 1min,and 95°C for 30s.

    The primer sequences used for qRT-PCR are presented in Supplementary Table S1.

    For comparison of the expression of inflammatory factors between the transgenic and wild-type groups of sheep under E.coli challenge,the cells were treated with E.coli at a multiplicity of infection(MOI)of 10 and incubated for 30min,and total RNA was extracted after first washing the cells three times with PBS.The mRNA expression levels were normalized to those of a housekeeping gene,GAPDH.The cells treated with S.aureus underwent the same procedure as the E.coli cells.The data were analysed using the comparative 2?ΔΔCTmethod.

    Inhibitor treatment

    For analysis of the interactions among TLR4,caveolaedependent endocytosis,and Src signalling in E.coli internalization and elimination,monocytes were pretreated for 2h with either 2μmol/L Tak242(MedChemExpress,Monmouth Junction,USA) —a TLR4 inhibitor,3μmol/L filipin—a caveolae-mediated endocytosis inhibitor(MedChemExpress),or 3μmol/L dasatinib(MedChemExpress)—an Src inhibitor.After treatment, the cells were used for subsequent experiments.

    Monocyte endocytosis assay

    E.coli were labelled with pHrodo from a pHrodo? Red E.coli kit(Thermo Fisher Scientific).Before the powder was diluted in PBS(pH 7.4)to form a working solution,the mixture was sonicated for 30 min in the dark.Monocytes were plated in a 6-well plate(Corning)at 1×106cells/well and incubated for 2 h at 37°C.The monocytes were then infected with pHrodo-labelled E.coli at a ratio of 10:1(bacteria:cells)in RPMI-1640 medium supplemented with 10% FBS.After centrifugation at 200×g for 2 min,the cells in the 6-well culture plates were further cultured for the indicated times.The assay was terminated at each time point by washing the cells three times with cold PBS,followed by the addition of 4% paraformaldehyde to each well.Subsequently,the cells were subjected to flow cytometry and microscopy studies.

    Flow cytometry

    The percentage of monocyte endocytosis was measured by the uptake of red fluorescent pHrodo Escherichia coli(Thermo Fisher Scientific).Briefly,1×106cells/well monocytes were treated with pHrodo-labelled E.coli at a ratio of 10:1(bacteria:cells)in a 6-well plate.After indicated times incubation,cells were washed and fixed with 4% paraformaldehyde then harvested in PBS pH 7.4.Samples were analysed by a flow cytometer(Beckman Coulter).Live cells were gated by forward scatter and side scatter area,singlets were gated by forward scatter area and forward scatter height.1×104cells were collected,the PE channel was used to detect pHrodolabeled E.coli in cells.The pHrodo threshold was set based on the sample of cells without pHrodo addition and applied to all samples.The percentage of monocyte endocytosis was the proportion of positive pHrodo cells in total cells.Dates were analysed with FlowJo software.

    Endosome pH

    Each sample had the same monocytes in the test wells and standard curve wells.Monocytes were infected with pHrodo-labelled E.coli for 10min in 96-well culture plates and then washed with cold PBS.Noninternalized pHrodo-labelled E.coli was washed away.In addition,this time was treated as the starting point(0min).In standard curve wells,monocytes were treated for 30min at a fixed pH(4.21,5.18,6.2,7.38,or 8.16)with PBS containing 1% Triton X-100.Monocytes in the test wells tested at a predetermined time.The time when noninternalized pHrodo-labelled E.coli was washed away was taken as time 0.Fluorescence values were read at intervals using an emission wavelength of 590nm.The cell luminescence value without pHrodo-labelled E.coli was used as the background control.The sample values were compared with standard curves obtained using each sample.

    Colocalization assay

    E.coli was heat-inactivated at 90°C for 30min.After being washed twice with 0.1mol/L NaHCO3(pH9.0),heatinactivated E.coli was incubated in 0.1mol/L NaHCO3containing 1mg/mL FITC(Sigma-Aldrich,Saint Louis,USA)for 1h at room temperature with gentle stirring.Monocytes were plated onto 8-well glass chamber slides at a concentration of 1×105cells/well and incubated for 2h at 37°C to allow the cells to adhere.The cells were infected with FITC-labelled E.coli at an MOI of 10 for 30 min.Before incubation at 37°C,the plates were centrifuged to synchronize endocytosis.The cells were washed three times with PBS after incubation and then fixed with 4% paraformaldehyde for 20min,followed by the addition of 1% Triton X-100 to make the cells permeable.The cells were immunostained with EEA1(ab2900,1:500;Abcam,Cambridge,UK)or LAMP1(ab67283,1:500;Abcam).Alexa Fluor 647-conjugated antirabbit antibody(Beyotime)was used for detection.

    Cathepsin B activity assay

    Monocytes were collected by centrifugation and lysed in 100μL of prechilled cell lysis without inhibitors(Beyotime)on ice for 10min.Then,the cells were centrifuged at top speed for 5min,and the supernatant was transferred to a new tube.The protein concentration of each extract was quantified by a BCA Protein Assay Kit(Beyotime).The amount of total protein used for detection was the same.Each sample was measured using a cathepsin B activity assay kit(Yuanmu,Shanghai,China)according to the manufacturer’s protocols.The OD was determined at 450nm.The sample value was obtained by the standard curve.

    Statistical analysis

    All data are shown as the mean±SD from no fewer than three independent experiments. The differences observed among samples were analysed with the independent-samples t-test and were considered statistically significant when P<0.05.

    Results

    Detection of transgenic sheep overexpressing TLR4

    The TLR4 expression vector(Fig.1a)was integrated into the genome of transgenic sheep bred during our previous study.Positive individuals were confirmed for use in the present study by Southern blotting.After digestion of the sheep genome with the restriction enzyme HindIII,the endogenous TLR4 fragment(4700 bp)was found in all sheep,while the exogenous TLR4 gene(2771 bp)was only identified in the transgenic sheep(Fig.1b).

    Monocytes are vital innate immune cells that play critical roles in the response to infection and inflammation through antigen presentation[32,33].Therefore,we isolated monocytes from ovine peripheral blood to determine the level of TLR4 expression in these cells.The monocyte markers CD14 and CD11b were detected on the cell membranes by antibody staining(Fig.1c),and TLR4 expression in the transgenic and wild-type sheep was compared at the transcription and protein levels(Fig.1d–f).The results showed that the mRNA and protein levels of TLR4 were significantly higher(P<0.05 and P<0.01,respectively)in the transgenic ovine monocytes than in the wild-type monocytes.

    TLR4 overexpression leads to increased internalization and killing of E.coli and promotes the activation of multiple TLR4-mediated pathways

    The monocyte-mediated internalization of bacteria is an important antimicrobial reaction[34].Here,the number of internalized E.coli in the TG and WT monocytes both increased with increasing infection time,although the levels of intracellular bacteria were significantly higher(P<0.01 for all time points)in the TG monocytes than in the WT monocytes when using an MOI of 10(Fig.2a).However,the numbers of internalized S.aureus in the TG and WT monocytes were not different at any infection time(Fig.S1A).Next,the monocytes were challenged for a fixed time with E.coli and then cultured for the indicated times(Fig.2b).The bacterial burden in the TG monocytes was significantly higher(P<0.01)than that in the WT monocytes when the endocytosis time was 15 min.Both groups were treated with a TLR4 inhibitor,TAK242,which yielded similar results between the two groups(P<0.01),in addition to a significant reduction in the number of bacteria carried by either group(P<0.01).However,these differences between the TG and WT groups were not statistically significant after an endocytosis time of 30min.The changing number of E.coli bacteria internalized by the cells over different endocytosis times can be attributed to the increased level of bacterial elimination by the monocytes with increasing endocytosis time(Fig.2c).The elimination capacity of the TG group was significantly higher than that of the WT group regardless of whether the monocytes were treated with TAK242(P<0.01 without inhibition,P <0.05 with inhibition).Furthermore,TAK242 significantly affected the bacterial killing capacity of both groups(P<0.01).

    Fig.2 TLR4 overexpression promotes the activation of multiple pathways and increased E.coli internalization and killing.a Different infection times with E.coli(MOI=10)and bacterial internalization was detected in transgenic(TG)and wild-type(WT)monocytes.b TG and WT monocytes were incubated with E.coli(MOI=10)at the same time(15min),the bacteria that were not internalized were washed away,and the monocytes were cultured further in the incubator.Bacterial levels in the monocytes were determined with a colony-forming unit(CFU)count,and the start of endocytosis(0min)was when the bacteria were first added.The effect of the TLR4 inhibitor,TAK242,was also tested.c The numbers of bacteria killed in(b)were calculated by subtracting the number of intracellular bacteria at 30min from the same number at 15min.d The mRNA expression levels of TLR4,the proinflammatory cytokines TNFα and IL-6,and the anti-inflammatory cytokine IFN-β were determined in monocytes by qRT-PCR before and after E.coli infection for 30min.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.DMSO,dimethyl sulfoxide(control);MOI,multiplicity of infection

    The bacteria-induced activation of TLR4 often leads to the production of multiple cytokines to protect against infection[35,36].To elucidate how the overexpression of TLR4 influences pathway activation,we examined the production of TNFα (tumour necrosis factor α)and IL-6(interleukin 6)by the TIRAP-MyD88-dependent pathway and the production of IFN-β (interferon β)by the TRAM-TRIF-dependent pathway by qRT-PCR during the early stages of infection.All cytokines were produced at significantly higher levels(P<0.05)in the TG group than the WT group after 30min of E.coli infection(Fig.2d).In the S.aureus infection experiment,TLR4 expression in the WT and TG groups did not change significantly after 30 min of incubation (MOI=10)(Fig.S1B).

    TLR4 overexpression promotes monocyte endocytosis

    To explore the effect of TLR4 overexpression on the ability of monocytes to endocytose bacteria,we used E.coli labelled with pHrodo.This unique fluorogenic dye is only incorporated into acidic cellular compartments and allows detection of these organelles by increasing the fluorescence intensity as the pH of the surroundings becomes more acidic.Endocytosis is a process accompanied by a rapid decrease in endosomal pH[15].Internalized bacteria labelled with pHrodo are fluorescent and easily observed by microscopy,while extracellular bacteria are not fluorescent in the medium and are therefore not visible[37].The results showed that the overexpression of TLR4 during the initial stages of bacterial infection(10 and 30 min)led to more monocytes undergoing internalization,and the fluorescence signals from these cells were more intense(Fig.3a).Flow cytometric analysis showed that the percentage of monocyte endocytosis of pHrodo-labelled E.coli in the TG group was significantly higher(P<0.05)than that in the WT group for both E.coli infection times(Fig.3b,c and Supplementary Fig.S2).After pretreatment with the TLR4 inhibitor,TAK242,this percentage was still significantly higher(P<0.05)in the TG group than in the WT group,and the inhibitory effect of TAK242 on the TG monocytes was stronger than that on the WT monocytes(Fig.3d and Supplementary Fig.S3).These results suggest that the overexpression of TLR4 enhances monocyte endocytosis of E.coli.

    Fig.3 TLR4 overexpression promotes monocyte endocytosis.a The endocytosis of pHrodo-labelled E.coli by monocytes in the transgenic(TG)and wild-type(WT)groups was observed by fluorescence microscopy.Scale bar:100μm.b Flow cytometric analysis of the percentage of monocyte endocytosis of the pHrodo-labelled E.coli after 10min and 30min of infection.c The effect of pretreatment with the TLR4 inhibitor TAK242 on monocyte endocytosis of pHrodolabelled E.coli.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.DMSO,dimethyl sulfoxide(control)

    TLR4 overexpression promotes caveolae formation

    Caveolae-mediated endocytosis is reported to be an extremely important pathway for realizing pathogen internalization [38],particularly towards E.col[39].CAV1 is a signalling protein associated with caveolae;its phosphorylation by the tyrosine kinase Src activates CAV1 and promotes caveolae formation[20,26].To investigate the role of caveolae-mediated endocytosis in the underlying mechanism of TLR4 overexpression promoting E.coli endocytosis,the key proteins involved in caveolae formation,Src and CAV1,as well as their phosphorylation levels,were detected during the early stages of E.coli infection.The results showed that the phosphorylation of Src and CAV1 in the TG group started earlier than that in the WT group(Fig.4a).Furthermore,at each time point,the levels of Src and CAV1 phosphorylation were significantly higher than those of the WT group.There were particularly significant differences in Src phosphorylation at 30min and CAV1 phosphorylation at 60min(P<0.01 for both;Fig.4b,c).However,the protein levels of Src and CAV1 were not significantly different between the two groups except for CAV1 at 10min(Fig.4d,e).These results suggest that the overexpression of TLR4 enhances Src and CAV1 phosphorylation,which can lead to increased levels of caveolae-mediated endocytosis during the initial stage of E.coli infection.

    Fig.4 TLR4 overexpression promotes the internalization of E.coli through caveolae-mediated endocytosis.a The activation of proteins involved in caveolae-mediated endocytosis signalling in transgenic(TG)and wild-type(WT)monocytes was detected by immunoblotting at different time points after E.coli infection.b,c The phosphorylation levels of Src and CAV1 were quantified based on immunoblotting.d,e The protein expression levels of Src and CAV1 were quantified based on immunoblotting.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.GAPDH was used for normalization

    Inhibition of caveolae-mediated endocytosis reduces the ability of TLR4-overexpressing monocytes to internalize and kill E.coli

    To better clarify the role of caveolae-mediated endocytosis in the antimicrobial activity of monocytes overexpressing TLR4,we performed experiments with various inhibitors.Prior to western blotting,TLR4-overexpressing monocytes were pretreated with either dasatinib(an Src inhibitor),filipin(a strong caveolae-mediated endocytosis inhibitor),or TAK242(a TLR4 inhibitor)and subjected to 30min of E.coli infection.The levels of phosphorylated Src and CAV1 in all treatment groups decreased(Fig.5a–c),especially those of p-Src in the dasatinib and TAK242 treatment groups(P <0.01)and p-CAV1 in the filipin treatment group(P<0.01).However,there were no significant differences in the Src and CAV1 protein levels following inhibitor treatment(Fig.5a,d,e).Flow cytometric analysis of the percentage of monocyte endocytosis of pHrodo-labelled E.coliin the untreated TLR4-overexpressing monocytes was compared with that of the dasatinib-and filipin-treated cells.Both inhibitors significantly reduced(P<0.01)the percentage of endocytosis(Fig.5f,g and Supplementary Fig.S4).The same results were observed for the intracellular E.coli CFU count(Fig.5h)and the numbers of killed E.coli based on the CFU count(Fig.5i).These results showed that the antimicrobial activity of the TLR4-overexpressing monocytes can be reduced by inhibitors of caveolae-mediated endocytosis.This finding suggests that caveolae-mediated endocytosis plays an important role in the antibacterial activity of monocytes overexpressing TLR4.

    Fig.5 Inhibition of caveolae-mediated endocytosis reduces TLR4-overexpressing monocyte endocytosis and kills E.coli.a Monocytes were pretreated with the control(DMSO)or various inhibitors for 2h,and after 30min of E.coli infection(MOI=10),the protein levels of Src,CAV1,p-Src,and p-CAV1 were determined by western blotting.b–e Quantification of the data in(a).GAPDH protein was used for normalization.f–g Flow cytometric analysis of the percentage of monocyte endocytosis of pHrodo-labelled E.coli(MOI=10)by the TLR4-overexpressing cells pretreated with DMSO or the inhibitors dasatinib(3μmol/L)or filipin(3μmol/L)prior to 30min of infection with pHrodo-labelled E.coli.h The quantity of E.coli internalized within monocytes after 30min of infection(MOI=10)was determined based on a colony-forming unit(CFU)count.i After 15min of incubation with E.coli(MOI=10),the bacteria that were not internalized were washed away,and the cells were cultured for an additional 15min in the incubator.The number of E.coli killed in the monocytes was calculated based on the difference in the CFU values at these two time points.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.DMSO,dimethyl sulfoxide;MOI,multiplicity of infection

    TLR4 overexpression promotes endosome acidification and maturation

    To elucidate the enhanced E.coli killing capacity of TG monocytes,we examined this issue from the perspective of endosome maturation.The function of the endosome network is to collect internalized cargoes and ferry them to their final destinations[40].In this process,endosomes undergo a multitude of changes,including a drop in luminal pH.Here,pHrodo-labelled E.coli was used to compare endosomal pH changes between the TG and WT groups.Within 1 h after infection,the endosomal pH of the TG group exhibited a steeper decline than that of the WT group(Fig.6a).Furthermore,the pH of the TG group was significantly lower than that of the WT group at all time points after 0 min(P<0.05 to P<0.01).When the TG cells were pretreated with the inhibitors dasatinib,filipin,and TAK242,all inhibitors were found to significantly reduce the acidification of the endosomes(P<0.01 for all inhibitors and time points after 0 min;Fig.6b).To further verify the influence of TLR4 overexpression on the maturation of the endosome,we used FITC-labelled E.coli to analyse colocalization with early endosome antigen 1(EEA1,a primary endosomal marker)or lysosomal associated membrane protein 1(LAMP1,a lysosomal marker).From the fluorescence images,it was observed that for both the EEA1 and LAMP1 markers,the TG group showed greater colocalization than the WT group(Fig.6c,d).These results demonstrated that the overexpression of TLR4 can promote endosome acidification and that this acidification is regulated by the Src,caveolae-mediated endocytosis,and TLR4 pathways.In addition,the colocalization experiment with bacteria and endosomal markers for different stages of maturation provides another perspective on how the overexpression of TLR4 promotes endosomal maturation.

    Fig.6 TLR4 overexpression promotes endosome maturation.a The endosomal pH values of transgenic(TG)and wild-type(WT)monocytes were assessed following infection with pHrodo-labelled E.coli(MOI=10).Fluorescence values were read at intervals using an emission wavelength of 590nm.Each sample had its own standard curves.The values were compared with standard curves obtained using corresponding sheep monocytes treated for 30min at a fixed pH(4.21,5.18,6.2,7.38,or 8.16)in phosphate-buffered saline containing 1% Triton X-100.The start of the experiment(0min)was when the bacteria were first added.b Effects of inhibitor treatment(2μmol/L TAK242,3μmol/L dasatinib,and 3μmol/L filipin)on the endosomal pH of the TG and WT monocytes.Dimethyl sulfoxide(DMSO)was used as the control.c Fluorescence images of FITC-labelled E.coli colocalization with the primary endosomal marker EEA1 in the TG and WT monocytes after 30min of infection.Scale bar:10μm.d Fluorescence images of FITC-labelled E.coli colocalization with the lysosomal marker LAMP1 in the TG and WT monocytes after 30min of infection.Scale bar:10μm.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.DAPI,cell nuclei stain

    TLR4 overexpression promotes cathepsin B activity

    Cathepsin B is a kind of pH-activated protease that is important in protein degradation[41].This molecule is present in endosomes,and changes in endosomal pH modulate cathepsin B activity[42].Cathepsin B activity was examined to further clarify the effect of TLR4 overexpression on the protein degradation of monocytes.In our results,the cathepsin B activity in the TG monocytes was significantly higher(P<0.01 for all time points except 0 min)than that in the WT monocytes when E.coli was infected using an MOI of 10.The levels in the TG group increased steeply with increasing infection time in an hour(Fig.7a).After 30min of infection with E.coli,the TG cells pretreated with the inhibitors dasatinib,filipin,and TAK242 had significantly reduced cathepsin B activity(P<0.01)(Fig.7b).

    Fig.7 TLR4 overexpression enhances cathepsin B activity.a The relative cathepsin B activity of transgenic(TG)and wild-type(WT)monocytes was assessed following infection with E.coli for different times(MOI=10).b Monocytes were pretreated with the control(DMSO)or various inhibitors for 2h,and after 30min of E.coli infection(MOI=10),cathepsin B activity was compared.All data are presented as the mean±SD,n≥3;*P<0.05,**P<0.01.DMSO,dimethyl sulfoxide;MOI,multiplicity of infection

    Discussion

    TLR4 is a pattern-recognition receptor that plays a key role in the resistance of host immune cells to Gramnegative bacteria.When TLR4 binds to its specific ligands,it triggers the activation of the innate immune response[43].Several studies have focused on the function of TLR4 in bacterial infection.TLR4-deficient mice are more susceptible to infection than control mice and have higher bacterial loads[17,44].TLR4-mutant enterocytes exhibit decreased sensitivity to LPS compared with wild-type cells[45].Mice overexpressing TLR4 show greater protection against Salmonella infection because of their improved control of bacterial growth in target organs and the increased expression of important immune response genes in their spleen[46].These results demonstrated that TLR4 has extraordinary functional importance in endocytosis and inflammatory regulation as part of the host defence.Mechanistic studies have shown that the overexpression of TLR4 can efficiently and rapidly activate the TIRAP-MyD88-dependent pathway,while also promoting the innate antiviral effect by activating the TRAM-TRIF-dependent pathway[30].In addition,during the internalization of E.coli,the expression levels of TLR4,MAPKs,and scavenger receptors were all higher in the TLR4-overexpressing transgenic sheep than in the wild-type sheep.The level of actin polymerization and the adhesive capacity of sheep monocytes are also higher in the transgenic sheep than the control sheep[31].However,the defence mechanisms involved in the protective effect of TLR4 overexpression in sheep against invading pathogens are still not fully understood.In this study,using monocytes isolated from the peripheral blood of transgenic sheep,the expression of TLR4 at the mRNA and protein levels was observed to be significantly higher than that in the WT monocytes.After E.coli infection,the mRNA level of TLR4 increased in both groups but remained significantly higher in the TG group.The production of TNFα and IL-6 along the TIRAP-MyD88-dependent pathway and the production of IFN-β by the TRAM-TRIF-dependent pathway were compared between the two groups,and the TG group showed significantly higher expression of these factors than the WT group.Since TLR4 is the only TLR that uses all four adaptors and activates both of these pathways[43],these two pathways are not activated simultaneously.Instead,the activation of these pathways is regulated by the location of TLR4.Higher activation of the TRAM-TRIF-dependent pathway indicates that more TLR4 is transferred from the cell surface to endosomes.Thus,the endocytosis pathway can activate the TRAM-TRIF-dependent pathway,which is illustrated by the TG group exhibiting higher levels of endocytosis than the WT group.This finding is consistent with other studies[11,47,48].This inference was validated by experiments using monocytes infected with E.coli followed by CFU counts.However,there was no difference in Gram-positive bacteria.Inhibition of the TLR4 pathway significantly reduced the endocytosis and killing capacity of monocytes specific to the Gram-negative bacteria.

    Endocytosis during the early stages of infection was further examined in the TG and WT monocytes using pHrodo-labelled E.coli.We found that while the percentage of monocyte endocytosis of pHrodo-labelled E.coli in the two groups increased with increasing infection time,the TG monocytes exhibited a higher percentage of endocytosis.Furthermore,the TLR4 inhibitor TAK242 reduced the percentage of internalization to a greater degree in the TG group.This finding may be attributable to TG monocytes having more TLR4 receptors on their surface,leading to more sensitive bacterial detection and more monocytes participating in bacterial internalization.

    Cells have evolved a variety of strategies to endocytose particles,solutes,and bacteria[49].TLR4 signalling is associated with receptor internalization and trafficking,and which endocytosis pathways are used during these processes is still not fully understood.The caveolae share a lipid composition with plasma membrane rafts and represent a subdomain that is stabilized by caveolin proteins.The formation of caveolae is highly complex and regulated by cellular processes[50].It has been noted that rafts act as potential sites for LPS to interact with CD14,and TLR4,TLR4 and accessory proteins can associate with plasma membrane rafts.The TLR4-raft association is stimulated under LPS[48].However,caveolae involvement in TLR4-mediated endocytosis has not been fully elucidated.Recent studies have shown that the deficiency of the protein LAPF(lysosome-associated and apoptosis-inducing protein containing pH and FYVE domains),which affects the TLR4 pathway,can impair the phagocytosis and killing of E.coli by influencing caveolae complex formation in macrophages of mice[21].Here,by detecting the phosphorylation of key proteins,we aimed to determine whether the higher endocytosis and killing capacity of the TG monocytes was caused by increased levels of caveolae-mediated endocytosis.We found that the TG group exhibited earlier phosphorylation of Src and CAV1 following infection,and the levels of phosphorylated Src and CAV1 in the TG group were higher than those in the WT group at the same time points post-infection.In addition,Src is required for Src-CAV1 complex formation and bacterial endocytosis.The inhibition of Src and caveolae-mediated endocytosis reduced the endocytosis and bactericidal capacity of the TG group,which further indicated that caveolaemediated endocytosis was implicated in the enhanced capacity of TG monocytes for antimicrobial defence.

    Acidification and maturation of endosomes promote the killing of intracellular bacteria[51,52].However,whether TLR4 causes endosome acidification is controversial[16,53].We next explored the acidification and maturation of endosomes to examine the enhanced killing of intracellular bacteria by the TG group.The TG group showed increased acidification of the endosome,and the internalized bacteria showed greater colocalization with both the primary endosomal marker EEA1 and the lysosomal marker LAMP1 than those of the WT group.Inhibitors of Src,caveolae-mediated endocytosis,and TLR4 all reduced the degree of acidification in the TG group.Cathepsin B activity as an indicator of protein degradation ability was also examined.The activity of cathepsin B is strongly affected by pH,and increased pH leads to a reduction in cathepsin B activity[54].Our results of cathepsin B activity showed the same trend as phagosome acidification.All the results may indicate that TLR4 and caveolae-mediated endocytosis regulate endosome acidification and maturation.

    Conclusions

    Our results provide novel evidence of the underlying antibacterial mechanisms of TLR4 in the innate immunity of sheep.TLR4 overexpression in monocytes enhances their internalization and killing of bacteria via caveolae-dependent endocytosis and promotes their ability to fight against pathogens during the earlier stages of infection;these are the first results from a large animal model.In addition,for the first time,the pH of endosomes was detected by artificially controlling the expression of TLR4,and it was proven that in the case of exogenous stimuli,the increase in TLR4 expression was correlated with the decrease in endosomal pH.A significant decrease in pH can inhibit the growth of bacteria and reflects the maturation of endosomes.Further experimental results indicate that TLR4 and caveolaemediated endocytosis regulate endosome acidification and maturation,and the overexpression of TLR4 can promote the maturation of endosomes and the killing of E.coli by enhancing the phosphorylation of Src and CAV1.Src and CAV1 can be used as potential targets to provide a theoretical basis for future breeding for disease resistance.

    Abbreviations

    TLR4:Toll-like receptor 4;E.coli:Escherichia coli;CAV1:Caveolin 1;TIR:Tollinterleukin1 receptor;TIRAP:Toll-interleukin 1 receptor domain containing adaptor protein;MyD88:Myeloid differentiation factor 88;TRAM:TRIF-related adaptor molecule;TRIF:TIR domain containing adaptor-inducing interferonβ;MD2:Myeloid differentiation protein 2;LPS:Lipopolysaccharide;MAPK:Mitogen-activated protein kinase;MAL:MyD88 adapter-like;PBS:Phosphate-buffered saline;FBS:Fetal bovine serum;FITC:Fluorescein isothiocyanate;CD14:Differentiation 14;DAPI:4′,6-Diamidino-2-phenylindole;SDS:Sodium dodecyl sulfate;TBST:Tris-buffered saline containing 0.1% Tween 20;S.aureus:Staphylococcus aureus;LB:Luria-Bertani;OD:Optical density;qRT-PCR:Quantitative reverse-transcription polymerase chain reaction;CFU:Colony-forming unit;TG:Transgenic;WT:Wild-type;DMSO:Dimethyl sulfoxide;TNFα:Tumour necrosis factor α;IL-6:Interleukin 6;IFN-β:Interferon β;MOI:Multiplicity of infection;EEA1:Early endosome antigen 1;LAMP1:Lysosomal associated membrane protein 1;LAPF:Lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains

    Supplementary Information

    The online version contains supplementary material available at https://doi.org/10.1186/s40104-021-00585-z.

    Additional file 1.Table S1.Gene-specific primers for quantitative realtime polymerase chain reaction(qRT-PCR).

    Additional file 2:Figure S1.(A)Bacterial internalization was detected in transgenic(TG)and wild-type(WT)monocytes after different periods of incubation with Staphylococcus aureus(MOI=10).(B)TLR4 expression of transgenic(TG)and wild-type(WT)monocytes before and after 30min of S.aureus infection(MOI=10).

    Additional file 3:Figure S2.Flow cytometric analysis of the percentage of monocyte endocytosis of pHrodo-labelled E.coli after 10 min and 30min of infection in the TG group and the WT group.

    Additional file 4:Figure S3.Flow cytometric analysis of the effect on endocytosis of pHrodo-labelled E.coli in both groups of monocytes pretreated with TAK242.

    Additional file 5:Figure S4.Flow cytometric analysis of the percentage of monocytes that endocytosed pHrodo-labelled E.coli(MOI=10)in the TLR4-overexpressing cells pretreated with DMSO or the inhibitors dasatinib(3μmol/L)or filipin(3μmol/L)prior to 30min of infection with E.coli.

    Acknowledgements

    Not applicable.

    Authors’contributions

    YL,YL and ZL designed the project and YL performed the experiments.Data curation and Formal analysis was by YL,YL and SD.Funding acquisition was by ZL.Written by YL.Discussion was carried out by YL,YL,YZ,XX,and RZ.Blood samples were collection by YZ,JZ and XZ.All authors contributed to the article and approved the submitted version.

    Funding

    This work was supported by National Science and Technology Major Project of China(2016ZX08008-003).

    Availability of data and materials

    The data analyzed during the current study are available from the corresponding author on reasonable request.

    Declarations

    Ethics approval and consent to participate

    The sample collection procedures were performed in accordance with a protocol from the Animal Welfare Committee of the China Agricultural University(Permit Number:XK662).

    Consent for publication

    All the authors read and agree to the content of this paper and its publication.

    Competing interests

    All authors declare that they have no conflict of interest.

    Author details

    1Beijing Key Laboratory for Animal Genetic Improvement,National Engineering Laboratory for Animal Breeding,Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture,College of Animal Science and Technology,China Agricultural University,Beijing,China.2Tianjin Institute of Animal Sciences,Tianjin,China.3State Key Laboratory of Agrobiotechnology,College of Biological Sciences,China Agricultural University,Beijing,China.4Institute of Laboratory Animal Sciences,Chinese Academy of Medical Sciences and Comparative Medicine Center,Peking Union Medical College,Beijing,China.

    Received:20 December 2020 Accepted:16 March 2021

    91精品国产国语对白视频| 2018国产大陆天天弄谢| 亚洲美女黄色视频免费看| 亚州av有码| 一级a做视频免费观看| 18禁裸乳无遮挡动漫免费视频| 婷婷色综合大香蕉| 国产伦精品一区二区三区视频9| 极品少妇高潮喷水抽搐| 免费黄网站久久成人精品| 精品人妻一区二区三区麻豆| 久久毛片免费看一区二区三区| 国产成人一区二区在线| 久久久久久久久久人人人人人人| 日韩人妻高清精品专区| 国产 一区精品| 国产精品秋霞免费鲁丝片| 亚洲美女视频黄频| 色吧在线观看| 日韩强制内射视频| 午夜91福利影院| 免费日韩欧美在线观看| 男人爽女人下面视频在线观看| 亚洲精品成人av观看孕妇| 晚上一个人看的免费电影| 亚洲成色77777| 免费人妻精品一区二区三区视频| 国产综合精华液| 在线免费观看不下载黄p国产| 日韩视频在线欧美| 亚洲欧美精品自产自拍| 一本—道久久a久久精品蜜桃钙片| 亚洲高清免费不卡视频| 亚洲国产成人一精品久久久| videosex国产| 久久久久久伊人网av| 91精品国产国语对白视频| 如何舔出高潮| 夫妻性生交免费视频一级片| 3wmmmm亚洲av在线观看| 国产极品天堂在线| 久久精品久久久久久久性| 午夜福利影视在线免费观看| 久久久国产一区二区| 视频区图区小说| 22中文网久久字幕| videosex国产| 久久久久久久久久成人| 免费人成在线观看视频色| 伊人久久国产一区二区| 国产亚洲一区二区精品| 一本久久精品| 国产成人免费无遮挡视频| 黄色怎么调成土黄色| 久久久久视频综合| 亚洲av男天堂| 国产精品女同一区二区软件| 精品一区二区三区视频在线| 国产乱来视频区| 伊人久久国产一区二区| 久久亚洲国产成人精品v| 人人妻人人澡人人爽人人夜夜| 三级国产精品欧美在线观看| 精品久久久久久久久亚洲| 亚洲精品色激情综合| 人人澡人人妻人| 国模一区二区三区四区视频| 精品国产国语对白av| av一本久久久久| av播播在线观看一区| 亚洲无线观看免费| 丝袜喷水一区| 一二三四中文在线观看免费高清| √禁漫天堂资源中文www| 亚洲天堂av无毛| 美女大奶头黄色视频| av视频免费观看在线观看| 久久久a久久爽久久v久久| 久久久精品区二区三区| 狂野欧美白嫩少妇大欣赏| 日韩免费高清中文字幕av| 丝袜美足系列| 亚洲色图综合在线观看| 九九久久精品国产亚洲av麻豆| 九色亚洲精品在线播放| 国国产精品蜜臀av免费| 久久久久久久久大av| 亚洲经典国产精华液单| 人妻一区二区av| 亚洲欧美色中文字幕在线| 国产片特级美女逼逼视频| 在线观看美女被高潮喷水网站| 91精品国产国语对白视频| 高清黄色对白视频在线免费看| 成人亚洲欧美一区二区av| 国产亚洲av片在线观看秒播厂| 黄色视频在线播放观看不卡| 亚洲av免费高清在线观看| 亚洲欧洲日产国产| 桃花免费在线播放| 国产极品天堂在线| 熟女av电影| 日韩成人伦理影院| 国产精品不卡视频一区二区| 美女中出高潮动态图| 大又大粗又爽又黄少妇毛片口| 亚洲精品国产av蜜桃| 中文字幕最新亚洲高清| 黄片无遮挡物在线观看| 波野结衣二区三区在线| 老熟女久久久| 久热久热在线精品观看| 免费大片18禁| 少妇猛男粗大的猛烈进出视频| 色哟哟·www| 桃花免费在线播放| 女性生殖器流出的白浆| 日韩av在线免费看完整版不卡| 久久久久久久久久久免费av| 欧美日本中文国产一区发布| 国产一区二区三区综合在线观看 | 国产在线一区二区三区精| 久久久久久人妻| 蜜桃久久精品国产亚洲av| 丁香六月天网| 亚洲精品国产av蜜桃| 超碰97精品在线观看| 国产精品久久久久久精品古装| 国产精品熟女久久久久浪| videossex国产| 亚洲国产av新网站| 欧美日韩成人在线一区二区| 2022亚洲国产成人精品| av.在线天堂| 日韩一本色道免费dvd| 夫妻性生交免费视频一级片| 十八禁高潮呻吟视频| 欧美97在线视频| 日韩,欧美,国产一区二区三区| 三级国产精品欧美在线观看| 最新中文字幕久久久久| 久久人人爽人人爽人人片va| 亚洲av成人精品一区久久| 伦精品一区二区三区| 国产极品粉嫩免费观看在线 | 国产色婷婷99| 欧美日韩精品成人综合77777| 男男h啪啪无遮挡| 新久久久久国产一级毛片| 成人毛片60女人毛片免费| 久久久久人妻精品一区果冻| av黄色大香蕉| 免费人妻精品一区二区三区视频| 91精品伊人久久大香线蕉| 在线观看人妻少妇| 久久99精品国语久久久| 午夜福利影视在线免费观看| 欧美+日韩+精品| 国产一区二区在线观看av| 在线 av 中文字幕| 欧美日本中文国产一区发布| 久久久久视频综合| 大香蕉97超碰在线| 亚洲国产欧美日韩在线播放| 国产 一区精品| av国产精品久久久久影院| 国产片特级美女逼逼视频| 人妻系列 视频| 天堂俺去俺来也www色官网| 人妻制服诱惑在线中文字幕| 午夜av观看不卡| 在线观看免费日韩欧美大片 | 韩国高清视频一区二区三区| 国产成人午夜福利电影在线观看| 老司机影院毛片| 亚洲熟女精品中文字幕| 久久99热这里只频精品6学生| 亚洲精品第二区| 91成人精品电影| 成年人免费黄色播放视频| 欧美精品一区二区大全| 日韩中文字幕视频在线看片| 久久 成人 亚洲| 欧美 日韩 精品 国产| 女性生殖器流出的白浆| 又大又黄又爽视频免费| 午夜免费鲁丝| 国产爽快片一区二区三区| 久久久久久久久久久免费av| 最新中文字幕久久久久| 卡戴珊不雅视频在线播放| 女的被弄到高潮叫床怎么办| 久久久欧美国产精品| 边亲边吃奶的免费视频| 99久久中文字幕三级久久日本| 成人18禁高潮啪啪吃奶动态图 | 色婷婷久久久亚洲欧美| 婷婷色av中文字幕| 午夜视频国产福利| 精品一区二区免费观看| 成人二区视频| 高清黄色对白视频在线免费看| 综合色丁香网| 80岁老熟妇乱子伦牲交| 日韩伦理黄色片| 中文乱码字字幕精品一区二区三区| 一个人看视频在线观看www免费| 爱豆传媒免费全集在线观看| 18禁动态无遮挡网站| 欧美xxⅹ黑人| 国产精品久久久久久av不卡| 亚洲人成网站在线观看播放| 亚洲国产欧美在线一区| 黑人巨大精品欧美一区二区蜜桃 | 成人亚洲精品一区在线观看| 免费播放大片免费观看视频在线观看| 中国国产av一级| 久久人人爽人人片av| 18+在线观看网站| 永久免费av网站大全| www.色视频.com| 18禁在线无遮挡免费观看视频| 性色av一级| 久久精品熟女亚洲av麻豆精品| 久久99一区二区三区| 美女国产视频在线观看| 亚洲国产精品一区三区| 日韩 亚洲 欧美在线| 一级毛片aaaaaa免费看小| 成人毛片60女人毛片免费| 亚洲一级一片aⅴ在线观看| 国精品久久久久久国模美| 在线观看www视频免费| 国产淫语在线视频| 国产成人精品久久久久久| 爱豆传媒免费全集在线观看| 高清欧美精品videossex| 纯流量卡能插随身wifi吗| 成年人免费黄色播放视频| 九色成人免费人妻av| 中文字幕亚洲精品专区| 国产极品天堂在线| 人妻少妇偷人精品九色| 伦精品一区二区三区| av福利片在线| 91久久精品国产一区二区成人| 能在线免费看毛片的网站| 久久精品国产亚洲网站| 纯流量卡能插随身wifi吗| 欧美老熟妇乱子伦牲交| 午夜日本视频在线| 国产精品久久久久久av不卡| 免费看光身美女| 各种免费的搞黄视频| 99久久综合免费| 亚洲综合色惰| 男男h啪啪无遮挡| 乱码一卡2卡4卡精品| 男女免费视频国产| 国产男女内射视频| 99九九在线精品视频| av在线app专区| 免费av不卡在线播放| 中国三级夫妇交换| 欧美精品亚洲一区二区| 久久毛片免费看一区二区三区| 免费av不卡在线播放| 国产精品国产av在线观看| 在线天堂最新版资源| 国产一区二区在线观看av| 久久韩国三级中文字幕| 中文字幕免费在线视频6| 欧美激情极品国产一区二区三区 | 午夜av观看不卡| 国产精品一区www在线观看| 免费高清在线观看日韩| 黄色毛片三级朝国网站| 十八禁网站网址无遮挡| 制服人妻中文乱码| 18+在线观看网站| 自拍欧美九色日韩亚洲蝌蚪91| 免费黄网站久久成人精品| av在线观看视频网站免费| 日韩亚洲欧美综合| 免费高清在线观看日韩| 热re99久久国产66热| 午夜福利,免费看| 夫妻午夜视频| h视频一区二区三区| 中国三级夫妇交换| 在线观看人妻少妇| 十分钟在线观看高清视频www| 久热久热在线精品观看| 亚洲熟女精品中文字幕| 亚洲av综合色区一区| 久久热精品热| 亚洲欧美一区二区三区国产| 久久久久久久久久久久大奶| 高清欧美精品videossex| videosex国产| 啦啦啦中文免费视频观看日本| 考比视频在线观看| 一级a做视频免费观看| 午夜福利视频在线观看免费| 国产极品粉嫩免费观看在线 | 天天躁夜夜躁狠狠久久av| 丝袜美足系列| 十八禁网站网址无遮挡| 少妇的逼好多水| 国产精品嫩草影院av在线观看| 新久久久久国产一级毛片| 免费久久久久久久精品成人欧美视频 | 久久女婷五月综合色啪小说| 亚洲精品久久午夜乱码| 不卡视频在线观看欧美| 校园人妻丝袜中文字幕| 国产在线视频一区二区| 国产高清有码在线观看视频| 久久鲁丝午夜福利片| 久久人人爽av亚洲精品天堂| a级片在线免费高清观看视频| 啦啦啦在线观看免费高清www| 亚洲av在线观看美女高潮| 少妇被粗大猛烈的视频| 亚洲国产成人一精品久久久| 午夜福利网站1000一区二区三区| 日本wwww免费看| 国产欧美日韩综合在线一区二区| 卡戴珊不雅视频在线播放| 国产亚洲av片在线观看秒播厂| 日韩av在线免费看完整版不卡| 中文字幕av电影在线播放| 久久久国产欧美日韩av| 美女大奶头黄色视频| 久久ye,这里只有精品| 亚洲人成网站在线播| 亚洲第一区二区三区不卡| 免费不卡的大黄色大毛片视频在线观看| 精品少妇黑人巨大在线播放| 亚洲天堂av无毛| 亚洲四区av| 肉色欧美久久久久久久蜜桃| 免费观看的影片在线观看| 黑丝袜美女国产一区| 亚洲高清免费不卡视频| 人体艺术视频欧美日本| 99久久精品国产国产毛片| 美女脱内裤让男人舔精品视频| 亚洲婷婷狠狠爱综合网| 精品国产露脸久久av麻豆| 91精品伊人久久大香线蕉| www.av在线官网国产| xxxhd国产人妻xxx| 国产欧美亚洲国产| 精品少妇内射三级| 插逼视频在线观看| 在现免费观看毛片| 精品人妻偷拍中文字幕| 人妻系列 视频| 日产精品乱码卡一卡2卡三| 51国产日韩欧美| 母亲3免费完整高清在线观看 | 男人操女人黄网站| 如何舔出高潮| 搡女人真爽免费视频火全软件| 在线亚洲精品国产二区图片欧美 | 亚洲国产毛片av蜜桃av| 久久久久视频综合| h视频一区二区三区| 国产精品三级大全| a级片在线免费高清观看视频| 制服人妻中文乱码| 91精品三级在线观看| 综合色丁香网| 伦理电影大哥的女人| 美女福利国产在线| 最近中文字幕2019免费版| av国产久精品久网站免费入址| 久久久国产精品麻豆| 男男h啪啪无遮挡| 免费av不卡在线播放| 午夜91福利影院| 国产有黄有色有爽视频| 精品一区二区三区视频在线| 亚洲精品视频女| 一区二区三区免费毛片| 国产精品99久久久久久久久| 人人澡人人妻人| 免费看不卡的av| 国产精品秋霞免费鲁丝片| 飞空精品影院首页| 美女主播在线视频| 国产亚洲一区二区精品| 男人操女人黄网站| 老熟女久久久| 大码成人一级视频| 亚洲人成网站在线观看播放| 国产精品一区二区在线不卡| 久久精品国产亚洲av涩爱| 校园人妻丝袜中文字幕| 波野结衣二区三区在线| 日本欧美国产在线视频| 亚洲精品视频女| 天堂俺去俺来也www色官网| 欧美日韩视频精品一区| 乱人伦中国视频| 99久久中文字幕三级久久日本| 久久久久久久亚洲中文字幕| 欧美少妇被猛烈插入视频| 另类亚洲欧美激情| 亚洲情色 制服丝袜| 99久久精品一区二区三区| av免费在线看不卡| 日本午夜av视频| 亚洲欧美清纯卡通| 考比视频在线观看| 日韩伦理黄色片| 国产在视频线精品| 欧美人与善性xxx| 精品午夜福利在线看| 999精品在线视频| 久久久久久久久久人人人人人人| 国产亚洲最大av| 国产色爽女视频免费观看| 亚洲av不卡在线观看| 日韩,欧美,国产一区二区三区| 亚洲精品亚洲一区二区| 一区二区三区四区激情视频| 乱码一卡2卡4卡精品| freevideosex欧美| 国内精品宾馆在线| 欧美激情国产日韩精品一区| 又粗又硬又长又爽又黄的视频| 一级黄片播放器| 国产男女超爽视频在线观看| 视频中文字幕在线观看| 亚洲欧美成人精品一区二区| 中文字幕av电影在线播放| 色网站视频免费| 免费观看的影片在线观看| 久久热精品热| 久热久热在线精品观看| 菩萨蛮人人尽说江南好唐韦庄| 国产日韩欧美在线精品| 成人影院久久| 黄色一级大片看看| 精品久久久精品久久久| 美女大奶头黄色视频| 人妻系列 视频| 看非洲黑人一级黄片| 国产亚洲精品第一综合不卡 | 国产av国产精品国产| 国产男女超爽视频在线观看| 欧美日韩av久久| 国产精品国产三级专区第一集| 国产精品蜜桃在线观看| 女人精品久久久久毛片| 国产视频首页在线观看| 亚洲美女搞黄在线观看| 3wmmmm亚洲av在线观看| 国产精品熟女久久久久浪| 欧美xxxx性猛交bbbb| 男女免费视频国产| 国语对白做爰xxxⅹ性视频网站| 国产高清不卡午夜福利| 高清不卡的av网站| 欧美+日韩+精品| 激情五月婷婷亚洲| 久久婷婷青草| 自拍欧美九色日韩亚洲蝌蚪91| 中文字幕人妻熟人妻熟丝袜美| 美女大奶头黄色视频| 日韩亚洲欧美综合| 我的老师免费观看完整版| 99久国产av精品国产电影| 日本黄大片高清| 国产精品熟女久久久久浪| 亚洲国产精品一区二区三区在线| 亚洲四区av| 九九在线视频观看精品| 男男h啪啪无遮挡| 色94色欧美一区二区| 啦啦啦视频在线资源免费观看| 亚洲在久久综合| 18禁在线播放成人免费| kizo精华| 亚洲欧洲精品一区二区精品久久久 | 精品久久国产蜜桃| 亚洲欧美色中文字幕在线| 亚洲综合色惰| 亚洲美女搞黄在线观看| 如日韩欧美国产精品一区二区三区 | 丰满少妇做爰视频| 久久鲁丝午夜福利片| 久久久久久久精品精品| 久久精品国产亚洲av涩爱| 亚洲美女搞黄在线观看| 大片电影免费在线观看免费| 免费看光身美女| 我要看黄色一级片免费的| 免费高清在线观看视频在线观看| 亚洲国产精品国产精品| 激情五月婷婷亚洲| 五月开心婷婷网| 嫩草影院入口| 夫妻性生交免费视频一级片| 日本免费在线观看一区| 午夜老司机福利剧场| 夫妻性生交免费视频一级片| 国产免费视频播放在线视频| 国产在线免费精品| 国精品久久久久久国模美| 国产在线免费精品| 91精品伊人久久大香线蕉| 国产白丝娇喘喷水9色精品| 在线观看免费高清a一片| 少妇高潮的动态图| 少妇猛男粗大的猛烈进出视频| 亚洲成人av在线免费| 少妇人妻精品综合一区二区| 日韩制服骚丝袜av| 国产成人午夜福利电影在线观看| 一区二区三区免费毛片| 最新的欧美精品一区二区| 久久精品国产鲁丝片午夜精品| 国产精品三级大全| 亚洲成色77777| 欧美xxxx性猛交bbbb| 少妇人妻 视频| 在线观看免费视频网站a站| 中文字幕av电影在线播放| 国产女主播在线喷水免费视频网站| 91精品国产九色| 亚洲综合色网址| 在线精品无人区一区二区三| 热99国产精品久久久久久7| 亚洲美女黄色视频免费看| 欧美精品高潮呻吟av久久| 午夜免费鲁丝| 我的老师免费观看完整版| 18禁动态无遮挡网站| 九色成人免费人妻av| 少妇高潮的动态图| 狂野欧美白嫩少妇大欣赏| 精品国产一区二区久久| 国产精品人妻久久久久久| 日韩欧美精品免费久久| 欧美日韩成人在线一区二区| 丝袜喷水一区| 人人妻人人添人人爽欧美一区卜| 国产精品久久久久久久电影| 性色av一级| 精品午夜福利在线看| 欧美日本中文国产一区发布| 国产精品一区www在线观看| 搡女人真爽免费视频火全软件| 亚洲人成网站在线观看播放| 午夜老司机福利剧场| 91国产中文字幕| 国产免费又黄又爽又色| 免费观看性生交大片5| 永久免费av网站大全| 亚洲人成网站在线播| 国产片内射在线| 高清午夜精品一区二区三区| 婷婷色av中文字幕| 2018国产大陆天天弄谢| 亚洲经典国产精华液单| 麻豆精品久久久久久蜜桃| 亚洲精品色激情综合| www.av在线官网国产| 51国产日韩欧美| 女人精品久久久久毛片| 91精品三级在线观看| 久久久久精品性色| 精品一区二区免费观看| 麻豆成人av视频| 免费日韩欧美在线观看| 视频区图区小说| 欧美老熟妇乱子伦牲交| 一本一本综合久久| av福利片在线| 蜜桃在线观看..| 欧美 亚洲 国产 日韩一| 少妇被粗大猛烈的视频| 18+在线观看网站| av不卡在线播放| 婷婷色综合大香蕉| 久久毛片免费看一区二区三区| 最近手机中文字幕大全| 日韩中字成人| 欧美变态另类bdsm刘玥| 中文字幕精品免费在线观看视频 | 爱豆传媒免费全集在线观看| 热99久久久久精品小说推荐| av在线播放精品| 插逼视频在线观看| 国产极品粉嫩免费观看在线 | 涩涩av久久男人的天堂| 精品午夜福利在线看| 日韩在线高清观看一区二区三区| av不卡在线播放| 亚洲欧洲国产日韩| 人人妻人人爽人人添夜夜欢视频| 曰老女人黄片| 九九久久精品国产亚洲av麻豆| 亚洲,欧美,日韩| 国产成人aa在线观看| 97超视频在线观看视频| 黄色配什么色好看| 少妇人妻精品综合一区二区| 五月玫瑰六月丁香| 久久女婷五月综合色啪小说| 成人二区视频| 精品一区在线观看国产|