| Objective: To establish acute Graft-Versus-Host Disease(a GVHD)mouse model and evaluate the therapeutic effects of rapamycin-conditioned dendritic cells on a GVHD.Methods: We established a mouse a GVHD model via allogeneic hematopoietic stem cell transplantation where donor cells from BALB/C mice(H-2Kd)were infused to the recipient C57BL/6 mice(H-2Kb).In vivo imaging model of dendritic cells(DCs)was built to monitor the in vivo distribution and migration of DCs ex vivo treated by rapamycin in a GVHD mouse.Ex vivo generated dendritic cells(DCs)were treated by rapamycin and were Adoptively infused into the a GVHD mouse with ex vivo generated rapamycin-treated DCs,whose inhibition effect on a GVHD was examined via the measurement of the changes of mice weight and survival time.Results: DCs treated by rapamycin exhibited a suppressed expression of allostimulatory markers of CD40,CD80,CD86 and MHCII,as well as chemokine receptors of CCR1(14.5 ± 1.4 vs.10.0 ± 2.1)and CCR5(12.7 ± 2.3 vs.7.2 ± 1.2),but not CCR7(7.8 ± 1.3 vs.6.2 ± 2.5).The bioluminescence imaging analysis showed that rapamycin conditioning did not affect DCs’ in vivo distribution in GVHD mouse,and rapamycin-conditioned DCs can also transfer to lymphonodus or spleen.Interestingly,the weight of a GVHD mouse infused with rapamycin-treated DCs decreased slower than control(-1.9 ± 1.2 vs.-2.4 ± 1.5,P < 0.05)and they survived longer than control(16.6 ± 6.7 vs.10.1 ± 5.5,P < 0.05),but their overall survival rate was not improved.Conclusion: The rapamycin could induce DCs towards the tolerant state by suppression the expressions of the surface markers,and the adoptively infused RAPA induced tolerant DCs(RAPA-t DCs)preserve the ability of homing to T cell-abundant region.In addition,the adoptively infused RAPA-t DCs could attenuate a GVHD to some extent but could not improve the overall survival rate.Objective: To investigate the expression level of costimulatory molecules,chemokine receptors,and inflammatory response of immature dendritic cells(im DCs)treated with Graphene oxide nanoparticles(GO)and the effect of GO on migration and homing ability of im DCs and to study its mechanism,to provide experimental data and theoretical basis for the design and application of graphene oxide-neuropeptide complex to induce toleogenic DCs to treat a GVHD.Methods: Three different sizes of GO were characterized.Each size of GO was co-incubated with mouse bone marrow-derived im DCs for 48 hours.The highest dose that did not affect cell proliferation was determined as application concentration of GO.Enzyme-linked immunosorbent assay(ELISA)was used to detect the cytokine secretion after GO treatment of im DCs,followed by the measurement of the expression of costimulatory molecule,chemokine receptor,and reactive oxygen species(ROS)on DCs via flow cytometry.GO-treated im DCs were collected and adoptively infused to the mouse through foot pads,then BLI was used to monitor the migration and homing of im DCs. Immunofluorescence staining was performed to identify the microfilaments and microtubules of im DCs among different groups.Result:(1)Three kinds of GO nanoparticles with different dimensions were characterized by transmission electron microscope(TEM),dynamic light scattering(DLS),Zeta potential meter and atomic force electron microscopy(AFM),all of which are single layer structure and have a thickness between 1-2 nm.They were named according to its size as S(small,about 50-200 nm),M(medial,about 200-500 nm),and L(large,about 500-1500 nm).(2)The cell survival rate of S-DCs,M-DCs and L-DCs was higher than 90% at 15.6 μg/ml concentration.Therefore,the working concentration of GO in this study was 15.6 μg/ml.(3)It was observed by TEM that GO of different sizes can be uptaken by DCs with different pattern of endocytosis in vitro.(4)The expression of CD40,CD80 and CD86 on Ctrl-DCs were(44.27 ± 3.46)%,(32.67 ± 1.70)% and(32.73 ± 1.52)%,those of S-DCs were(53.43 ± 0.55)%,(34.93 ± 1.01)%,and(43.83 ± 4.01)%,those of M-DCs were(54.43 ± 2.48)%,(45.10 ± 4.19)%,and(47.97 ± 0.85)%,those of L-DCs were(57.13 ± 2.70)%,(46.93 ± 0.86)%,and(49.97 ± 0.76)%,indicating that GO upregulated the expression of costimulatory molecules CD40,CD80,and CD86 on im DCs,P < 0.05.(5)IL-6 secreted by S-DCs,M-DCs,and L-DCs(42617.02 ± 341.24 pg/m L,51709.04 ± 10.78 pg/m L,and 48080.82 ± 4856.53 pg/m L)were higher than those of Ctrl-DCs(37361.91 ± 5357.26 pg/m L),IL-1β secreted by S-DCs,M-DCs,L-DCs(1683.46 ± 74.01 pg/m L,1655.18 ± 24.74 pg/m L,1424.51 ± 95.88 pg/m L)were all lower than those of Ctrl-DCs(1958.28 ± 81.75 pg/m L),TNF-α secreted by S-DCs and L-DCs(75771.43 ± 1924.05 pg/m L and 73084.24 ± 883.93 pg/m L)were lower than those of Ctrl-DCs(81662.73 ± 2249.19 pg/m L),and IL-12p70 secreted by S-DCs,M-DCs,and L-DCs(66.67 ± 2.01 pg/m L,57.54 ± 8.99 pg/m L,and 64.38 ± 5.97 pg/m L).were lower than those of Ctrl-DCs(80.74 ± 4.11 pg/m L),P < 0.05.(6)The expression of CXCR4 of S-DCs and L-DCs(35.47 ± 1.92,31.48 ± 1.01)% was significantly higher than that of Ctrl-DCs(24.94 ± 0.65)%,and the expression of CCR7 of S-DCs,M-DCs,and L-DCs(20.00 ± 2.53,13.04 ± 1.89,14.43 ± 3.71)% was significantly higher than Ctrl-DCs(10.45 ± 1.89)%,P < 0.05.(7)In the padfood migration model,the percentage of S-DCs,L-DCs,M-DCs and Ctrl-DCs migrating to PLN at 48 h was(0.17 ± 0.02 vs.0.12 ± 0.06 vs.0.11 ± 0.05 vs.0.07 ± 0.01),and the percentages of those at 72 h were(0.24 ± 0.05 vs.0.16 ± 0.09 vs.0.15 ± 0.05 vs.0.08 ± 0.02),indicating that the order of migration rates was S-DCs>L-DCs.>M-DCs>Ctrl-DCs,P < 0.05.(8)The results of confocal microscopy showed that the GO-treated im DCs had higher ductility and adhesion,and a large number of filoform protrusion were radially around them,especially S-DCs.Cell scaffolds variability of Ctrl-DCs was poorer and lacked adhesion between cells.(9)Flow cytometry was used to detect the production of ROS in different GO treated DCs: The average fluorescence intensities of ROS produced by S-DCs,M-DCs and L-DCs were(274.52 ± 13.44,244.51 ± 7.78,243.53 ± 27.58),all of which were significantly higher than that of Ctrl-DCs(165.51 ± 10.62),P <0.05.Conclusion: GO can down-regulate the expression of CD40,CD80 and CD86 on im DCs.IL-6 secretion of im DCs was up-regulated,whereas the IL-1β,TNF-α and IL-12p70 secretion of im DCs was inhibited by GO.Also,GO significantly up-regulated the expression of CXCR4 and CCR7 on im DCs.In vivo imaging showed that the rate of GO-treated DCs migrating from padfood to the axillary lymph nodes was approximately 1.5-3 times higher than that of the Ctrl-DCs.The rearrangement of microfilaments and microtubules,enhanced adhesion of GO-DCs were observed through confocal microscope.Obviously,GO can promote the production of reactive oxygen species of im DCs.In conclusion,GO enhanced the production of ROS on DCs to enhance rearrangement and adhesion of DCs,and ultimately promoted the migration and homing ability of im DCs.Objective: To establish in vivo imaging a GVHD mouse model(BALB/C→C57BL/6J)using BALB/C(H-2Kd)as donor and C57BL/6J(H-2Kb)as receptor.And to design and synthesize neuropeptides-graphene oxide complexs,by which tolerogenic dendritic cells(t DCs)were induced,then t DCs were infused intravenously to a GVHD model mouse to evaluate the inhibiting effect of t DCs on a GVHD.Methods: Enzyme-linked immunosorbent assay(ELISA)was used to detect cytokine secretion of im DCs treated with four different neuropeptides with low-dose LPS,through which UCN(one of those four neuropeptides)was selected and the pro-inflammatory cytokines secreted by DCs treated with UCN-GO complexes were detected.Flow cytometry was used to detect the expression of costimulatory molecules;CFSE-labeled T-cells derived from BALB/C mice,were mixed with UCN-DCs or Ctrl-DCs for 72 h,then the proliferation level of them was measured through flow cytometry.UCN-DCs were collected and adoptively infused to the mice via footpad,then BLI was used to monitor the distribution,migration,and homing pattern of DCs in vivo.UCN-GO-DCs were adoptively intravenously infused to a GVHD mouse model,the proliferation of firefly luciferase reporter gene-labeled T cells in a GVHD mouse was observed to investigate the inhibition effect of a GVHD.Result:(1)The pro-inflammatory cytokines IL-12p70,IL-6,IL-1β,and TNF-α serected by DCs treated with four neuropeptides UCN,VIP,α-MSH,and AM were compared within low-dose LPS stimulation.The concentration of UCN-DCs of IL-12p70(63.55 ± 9.19 pg/m L vs.102.92 ± 10.78 pg/m L),IL-6(2386.76 ± 524.44 pg/m L vs.27370.14 ± 1640.17 pg/m L),IL-1β(1248.70 ± 34.33 pg/m L vs.1574.46 ± 37.39 pg/m L)and TNF-α(27568.09 ± 2706.38 pg/m L vs.35040.75 ± 892.64 pg/m L)were lower than those of LPS-DCs,P < 0.05.Therefore,UCN was chose to induce t DCs.(2)The expression of costimulatory molecules CD40(55.03 ± 3.87 vs.65.38 ± 1.62)%,CD80(70.38 ± 2.30 vs.77.10 ± 2.56)%,and CD86(70.30 ± 2.37 vs.73.90 ± 3.49)% of UCN-DCs were down-regulated,P < 0.05.(3)Proliferation percentage of CFSE-labeled T cells in UCN-DCs group(22.89 ± 2.49 vs.48.10 ± 1.53)%,(1.92 ± 1.42 vs.59.81 ± 1.86)% and(5.94 ± 0.77 vs.35.40 ± 2.20)% were lower than those in the Ctrl-DCs group,when DCs were mixed with T cells at 2:1,4:1 and 8:1,P < 0.05.(4)7.8 μg/ml was selected as the cell viability of UCN-S-DCs and UCN-L-DCs was higher than 90% at this concentration.(5)The coupling efficiency of UCN with GO-S was higher than that of α-MSH,VIP and AM(89.33 ± 1.53 vs.76.00 ± 2.65 vs.47.33 ± 1.15 vs.36.00 ± 2.01)%,the coupling efficiency with GO-L was also higher than that of the other groups(86.33 ± 1.53 vs.55.67 ± 0.58 vs.35.33 ± 2.08 vs.32.67 ± 1.73)%,P < 0.05.(6)Within low dose LPS stimulation,the concentration of pro-inflammatory cytokines secreted by UCN-S-DCs and UCN-L-DCs was lower than that of UCN-DCs;IL-12p70(61.85 ± 4.99 pg/m L vs.61.03 ± 0.52 pg/m L vs.68.99 ± 3.91 pg/m L)、IL-6(19935.66 ± 1286.64 pg/m L vs.22733.32 ± 5396.41 pg/m L vs.30440.11 ± 1655.73 pg/m L)、IL-1β(1395.66 ± 62.12 pg/m L vs.1394.44 ± 62.12 pg/m L vs.1566.65 ± 13.82 pg/m L)、TNF-α(64292.69 ± 1278.26 pg/m L vs.61986.79 ± 2537.94 pg/m L vs.71771.30 ± 883.27 pg/m L),P < 0.05.(7)In vivo imaging software was used to analyze the percentage of DCs migrated from footpad to PLN: the percentages of Ctrl-DCs that migrated to PLN at 24 h,48 h,and 72 h were(0.04 ± 0.02),(0.07 ± 0.02),(0.09 ± 0.02),the percentages of UCN-DCs that migrated to PLN at 24 h,48 h,and 72 h were(0.06 ± 0.03),(0.10 ± 0.05),(0.11 ± 0.01);the percentages of UCN-S-DCs that migrated to PLN at 24 h,48 h,and 72 h were(0.10 ± 0.06),(0.11 ± 0.02),(0.15 ± 0.02);the percentages of migration to PLN at 24 h,48 h,and 72 h were(0.22 ± 0.07),(0.23 ± 0.07),(0.23 ± 0.04).Among them,the percentages of UCN-L-DCs migrated to PLN at 24 h,48 h,and 72 h were higher than those of UCN-DCs and Ctrl-DCs,and the percentages were higher than UCN-S-DCs at 72 h.The percentages of UCN-S-DCs migrating to PLN at 48 h and 72 h were higher than that of UCN-DCs and Ctrl-DCs,indicating that the order of migration rates was UCN-L-DCs>UCN-S-DCs>UCN-DCs>Ctrl-DCs/S-DCs/L-DCs,P < 0.05(8)In vivo imaging a GVHD mouse model was used to monitor the inhibiting effect of UCN-DCs on T cell proliferation: The total fluorescence intensity in each group of mice was lower than that of BM + Tc group(8.18 ′ 108 ± 3.06 ′ 107).On the sixth day after transplantation,the total fluorescence intensity in the UCN-DCs,UCN-S-DCs,and UCN-L-DCs groups was(1.28 ′ 107 ± 4.00 ′ 105)and(2.33 ′ 107 ± 2.25 ′ 106),which means that the inhibiting effect of UCN-DCs on T cell proliferation was stronger than that of Ctrl-DCs,and the inhibiting effect of UCN-S-DCs and UCN-L-DCs on T cell proliferation was stronger than that of UCN-DCs,P < 0.05.In other words,the loading by GO of UCN improve the inhibiting effect on T cell proliferation of UCN.Conclusion: UCN inhibited the secretion of pro-inflammatory cytokines of DCs significantly more than the other three neuropeptides,which maked UCN to be chose to induce t DCs.Further analysis revealed that UCN significantly inhibited the expression of costimulatory molecules CD40,CD80,and CD86 on im DCs,suggesting that UCN-DCs have reduced maturation of im DCs,and that their ability to activate T lymphocytes may decrease.CFSE-T cell proliferation experiments confirmed that UCN-DCs can induce T cell silencing and inhibit their proliferation.Using GO to highly efficient(89% coupling efficiency)couple with UCN on a concentration of 7.8 μg/ml that does not affect the proliferation of DCs to treat im DCs,whose cytokine levels were analyzed and confirmed that the loading by GO increased the effect of UCN to inhibit the cytokine secretion of DCs.Subsequent footpad infusion model and in vivo imaging model data show that UCN-GO can promote the local migration ability of DCs,and the effective inhibition of the proliferation of T cells in a GVHD mice by UCN-GO was initially proved. |
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