肺巨噬细胞移植治疗

1、肺巨噬细胞移植治疗肺巨噬细胞移植治疗(PMT)Pulmonary MacrophageTransplantation Therapy专业：细胞生物学姓名：李倩倩导师：毕秀丽 hPAP遗传性肺泡蛋白质沉积症简介 基因治疗 摘要 实验方法 实验过程和结果阐述 相关基因治疗案例 hPAPHereditary pulmonary alveolar proteinosis:即hPAP,遗传性肺泡蛋白质沉积症 纤维支气管镜活检病理学检查示肺泡腔内充满PAS阳性的粗颗粒状物质,肺泡灌洗液可见大量无定形的碎片,常伴PAS染色阳性的巨噬细胞。 在hPAP中，肺泡充满了表面活性物质，肺生成这种物质来降低表面张力，

2、保持肺泡开放状态。 hPAP患儿具有CSFR2RA或CSFR2RB突变。这些突变降低了肺泡巨噬细胞清除这些孩子肺部表面活性物质的能力。 表面活性物质累积于肺部，填满肺泡会造成呼吸困难或呼吸衰竭。对于这些孩子当前唯一的治疗方法就是在全身麻醉情况下完成侵袭性的洗肺程序。但其效应是暂时的，必须频繁重复操作，给受累患儿造成生活质量问题。GM-CSF：粒细胞集落刺激生物因子，能够刺激骨髓细胞对肺泡细胞的胞饮以及对面面活性物质的降解。GM-CSF受体即CSFR的缺陷或者GM-CSF抗体均可导致hPAP。 GM-CSF的受体为CSFR,CSFR由和和链链组成，组成， 编码编码和和的基因分别为的基因分别为cs

3、f2racsf2ra和和csf2rbcsf2rb。csf2racsf2ra和和csf2rbcsf2rb突变突变 CSFRBCSFRB表达缺陷表达缺陷 阻断阻断GM-CSFGM-CSF信号转导信号转导 巨噬细胞对肺泡表面蛋白清除能力下降巨噬细胞对肺泡表面蛋白清除能力下降 hPAP hPAP也就是说，也就是说，hPAPhPAP患者肺泡血清中患者肺泡血清中GM-CSFGM-CSF升升高，而高，而GM-CSFGM-CSF抗体为阴性抗体为阴性 基因治疗简介基因治疗（gene therapy）是指将外源正常基因导入靶细胞，以纠正或补偿因基因缺陷和异常引起的疾病，以达到治疗目的。也就是将外源基因通过基因转移

4、技术将其插入病人的适当的受体细胞中，使外源基因制造的产物能治疗某种疾病。基因治疗的特异性强，其药物本质为DNA，在体内表达为蛋白质，且基因治疗作用有持续性。基因治疗的策略有基因替代，基因修正，基因抑制，基因增强和免疫调节。 摘要：以往用骨髓移植(BMT)来治疗hPAP小鼠模型，会破坏现有骨髓。最近发现定居巨噬细胞可以自我维持，不需来自骨髓的细胞直接再生。于是作者想到测试新型巨噬细胞移植疗法。结果表明，自然的健康巨噬细胞和基因矫正巨噬细胞同样能够很好地发挥作用纠正小鼠的hPAP。在小鼠基因Csf2rb丧失表达、模拟hPAP的小鼠中，研究人员利用一种病毒载体将矫正Csf2rb基因传递到取自动物的异

5、常肺泡巨噬细胞中。随后将基因矫正细胞直接灌注到小鼠的肺部。这种方法使得疾病相关生物标记物正常化，至少在一年内降低了疾病死亡率。实验方法实验方法 支气管肺泡灌洗液和细胞收集、处理支气管肺泡灌洗液和细胞收集、处理 ELISA ,Quantitative RT-PCRELISA ,Quantitative RT-PCR Bone marrow derived macrophages Bone marrow derived macrophages (BMDMs)(BMDMs) Colony forming cell (CFC) assayColony forming cell (CFC) assay集

6、落形成集落形成实验实验 Surfactant Clearance AssaySurfactant Clearance Assay Pulmonary macrophage transplantation Pulmonary macrophage transplantation (PMT)(PMT) Flow cytometryFlow cytometry流式细胞术流式细胞术 STAT5 phosphorylation index assaySTAT5 phosphorylation index assay Western blottingWestern blotting Microarray

7、analysisMicroarray analysis微阵列分析微阵列分析 Localization of PMT-derived cells Localization of PMT-derived cells after transplantationafter transplantation Lentiviral vectorsLentiviral vectors（慢病毒载体）慢病毒载体）, and , and differentiation and expansion of differentiation and expansion of macrophagesmacrophages实验

8、过程简介三种小鼠模型1）WT mice(wind type) 2)KO mice(Csf2rb-/-) 3)KO+PMT mice(pulmonary macrophage transplantation)WT HSPCs isolated expanded differentiated into macrophagestransplantationhPAP markers： 1）PAS染色阳性 ORO染色阳性2）BAL turbiditySP-Dconcentration3）BAL中，GM-CSF M-CSF CSF1 MCR1 mRNA for PU1, PPAR ABCG1 结果结果 K

9、O小鼠可以作为人类hPAP患者的模拟生物(a) Typical lung pathology and identical pulmonary histopathology in aKO mouse. PAS stain.(b) Photographs of ilkyappearing BAL from a 14 month-old KO mouse and normal-appearing BAL from an age-matched WT mouse(d) BAL fluid biomarkers of hPAP(GM-CSF, M-CSF, and MCP-1) in 4 month-o

10、ld KO mice(e) Alveolar macrophage biomarkers (PU.1, Pparg, Abcg1 mRNA) arereduced in 4 month-old KO(f)(g) Progressive increase in BAL turbidityBAL fluid,GM-CSF level in KO mice (h) GM-CSF bioactivity in BAL fluid from 10 month-old KO or WT mice Result: We validated KO mice as a model of human hPAP b

11、y demonstrating they had the same clinical,physiological, histopathological, and biochemical abnormalities, disease biomarkers, natural historyas children with hPAP3. Macrophage characterization after PMT(a-b) Photomicrographs of WT BMDMs prior to transplantation phase-contrast (a) or DiffQuick stai

12、ning (b) (c) Flow cytometry evaluation of cell-surface phenotypic markerson WT BMDMs before PMT.(d)(d)Photographs ofPhotographs of methylcellulose cultures of methylcellulose cultures of Lincells from bone marrow Lincells from bone marrow and BMDMs and typical and BMDMs and typical coloniescolonies

13、(e) Colony counts of BFU-E, (e) Colony counts of BFU-E, CFU-GEMM andCFU-GEMM andCFU-GM showing BMDMs CFU-GM showing BMDMs contained 0.005% CFU-GM contained 0.005% CFU-GM and no BFU-E or CFU-GEMMand no BFU-E or CFU-GEMMprogenitors, corresponding to progenitors, corresponding to 93 CFU-GM per dose of

14、93 CFU-GM per dose of BMDMs administered (n=3BMDMs administered (n=3d e t e r m i n a t i o n s p e r d e t e r m i n a t i o n s p e r condition). condition). (f-g) Evaluation of surfactant clearance capacity.(f-g) Evaluation of surfactant clearance capacity.These results demonstrated the cells use

15、d for PMT were highly purified, mature macrophages capable of surfactant clearance.Efficacy of PMT of WT macrophagesEfficacy of PMT of WT macrophagesTo determine the therapeutic potential of PMT, KO mice received WT (Csf2rb+/+) BMDMs by PMT once (Fig. 1a). One year later, PMT-derived CD131 + BAL cel

16、ls were present (Fig.1b), alveolar macrophages expressed Csf2rb(c) Appearance of BAL fluid(c) Appearance of BAL fluid (left) or sediment (right). (left) or sediment (right). (b)Representative cytology of (b)Representative cytology of BAL obtained one year afterBAL obtained one year after PMT after s

17、taining with PAS PMT after staining with PAS or oil-red-O (ORO)or oil-red-O (ORO)PMT nearly completely resolved theabnormal pulmonary histopathology (c)Representative photomicrog r aphs of (c)Representative photomicrog r aphs of PASstained whole-mount lung sections one year PASstained whole-mount lu

18、ng sections one year after PMTafter PMTPMT nearly completely resolved theabnormal pulmonary histopathology (d) Lung histology after (d) Lung histology after staining with H&E, PAS,staining with H&E, PAS,Masson trichrome (MT), Masson trichrome (MT), or surfactant protein B or surfactant protein B (SP

19、-B)(SP-B)(e) BAL turbidity and SP-D concentration. (f) BAL biomarkers. ResultThese results demonstrate PMT had a highly efficacious and durable therapeutic effect on the primary pulmonary and secondary systemic manifestations of hPAP in KO mice.Macrophage engraftment efficiency is significantly diff

20、erent compared to untreated WT m i c e ( M a n n -Whitney Rank Sum Test, P0.001). is significantly different compared to untreated KO mice not significantly s i g n i f i c a n t l y different compared to untreated KO mice P=0.133.Neither significantly affected efficacy in the range evaluated and on

21、e dose of2 million cells was used for PMT in the remaining studies.To determine if WT macrophages had a survival advantage over KO macrophage we measured GM-CSF bioactivity in BAL fluid and found it was detectable in KO but not WT mice WT macrophages had increased WT macrophages had increased surviv

22、al/proliferationsurvival/proliferationcompared to KO macrophages compared to KO macrophages in vitro in vitro (Fig. 2a) and (Fig. 2a) and accumulated to greater numbers after PMT in KO mice accumulated to greater numbers after PMT in KO mice than in WT mice (Fig. 2b and Extended Data Fig. 3d)than in

23、 WT mice (Fig. 2b and Extended Data Fig. 3d)(b) Quantification of GFP+ BAL cells 2 months after PMT (b) Quantification of GFP+ BAL cells 2 months after PMT of Lys-MGFP BMDMs into WT (n=3) or KO (n=6) mice. of Lys-MGFP BMDMs into WT (n=3) or KO (n=6) mice. (d) GFP+ cells in BAL cells from WT or KO mi

24、ce 2 monthsafter PMT of Lys-MGFP BMDMs.PMT of WT LysMGFP knock-in mouse25 BMDMs into KO mice followed by Ki67 immunostaining revealed PMT-derived cells replicated in vivo (Extended Data Fig. 3e-g). Ki67是与增殖相关的核抗原，Ki67标记G0期(非增殖期)以外的细胞，Ki67阳性细胞比例越高，处于增殖时期的细胞比例越高，肿瘤恶性程度越高。GFPKi67GFP+Ki67(e) Macrophage

25、replication after PMT. KO mice received Lys-MGFP BMDMs by PMT and paraffin-embedded lung was immunostained for Ki67 one month or one year later(c)The percentage of Ki67+PMT-derived alveolar macrophages was 32.2 _+ 6.05% one month after PMT and declined to 11.29 _+ 2.2% by one year (Fig. 2c)(f) Ki67

26、staining of BAL cells froman untreated WT mice To further define survival advantage, we evaluated the engraftment kinetics after one PMT of WT BMDMs in KO mice. CD131 + cells increased steadily from zero to 69.0?2.5% of BAL cells(Fig. 2d) (e) a smooth decline in pulmonary GM-CSF to near normal (f) B

27、AL turbidity declined with the increase inCD131+ alveolarmacrophages.One year after PMT, CD131 + cells were present , One year after PMT, CD131 + cells were present , Csf2rb Csf2rb protein was detectable in alveolar macrophages, and protein was detectable in alveolar macrophages, and Csf2rb Csf2rb m

28、RNAmRNA in BAL cells from PMT-treated KO mice was in BAL cells from PMT-treated KO mice was only slightly less than in WT and undetectable in only slightly less than in WT and undetectable in untreated KO BAL cells (Fig. 2g). numbers ofuntreated KO BAL cells (Fig. 2g). numbers of CD131 + CD131 + alv

29、eolar macrophagesalveolar macrophages in PMT-treated KO and untreated in PMT-treated KO and untreated WT mice wereWT mice were similar similar one year after PMT (Fig. 2h). one year after PMT (Fig. 2h).These results 1) WT macrophages had a selective survival advantage over KO macrophages, 2)WT macro

30、phages after PMT into KO mice They proliferated in vivo at a rate that slowed over time synchronous with reduction in pulmonary GM-CSF, replaced dysfunctional K O a l v e o l a r m a c r o p h a g e s3 ) T h e n u m b e r s o f C D 1 3 1 + , G M - C S F responsive alveolar macrophages similar to WT

31、mice.Macrophage characterization after PMTMacrophage characterization after PMT( h )( h ) L o c a l i z a t i o nL o c a l i z a t i o n o f o f macrophages after PMT macrophages after PMT of Lys-MGFP BMDMs into of Lys-MGFP BMDMs into KO mice and after CD68 KO mice and after CD68 immunostaining,DAPI

32、 immunostaining,DAPI s t a i n i n g , a n d s t a i n i n g , a n d fluorescence microscopy fluorescence microscopy to detect CD68+/GFP+ to detect CD68+/GFP+ c e l l s P M T d e r i v e d c e l l s P M T d e r i v e d m a c r o p h a g e s o r m a c r o p h a g e s o r CD68+/GFPcells (i.e., CD68+/G

33、FPcells (i.e., n o n - P M T - d e r i v e d n o n - P M T - d e r i v e d macrophages).macrophages). CD68+/GFP+ revealed 88.9 +_ 0.87% were intra-alveolar CD68+/GFP+ revealed 88.9 +_ 0.87% were intra-alveolar and 11.1 _+0.87% were interstitial and 11.1 _+0.87% were interstitial GFPimmunohistochemic

34、al staining was done to eliminate GFPimmunohistochemical staining was done to eliminate potential interference from autofluorescence and potential interference from autofluorescence and confirmed these results; 90.5_+1.1% PMT-derived confirmed these results; 90.5_+1.1% PMT-derived macrophages were i

35、ntra-alveolar and 9.4_+ 1.1% were macrophages were intra-alveolar and 9.4_+ 1.1% were interstitial (Fig. 3a-b )interstitial (Fig. 3a-b )GFP+ cellsGFP+ cellsL o c a l i z a t i o n o f G F P + L o c a l i z a t i o n o f G F P + macrophages to intra-alveolarmacrophages to intra-alveolarLocalization w

36、as done in WT Lys-MGFP or KO mice and Localization was done in WT Lys-MGFP or KO mice and PMT-treated KO mice by flow cytometry to detect the PMT-treated KO mice by flow cytometry to detect the percentage of GFP+ cells one year after PMT (Extended percentage of GFP+ cells one year after PMT (Extende

37、d Data Fig. 4a-b) and by PCR amplification of Lys-MGFP Data Fig. 4a-b) and by PCR amplification of Lys-MGFP transgene-specific DNA (Extended Data Fig. 4c),transgene-specific DNA (Extended Data Fig. 4c),PMT-derived cells were PMT-derived cells were present in the lungs but present in the lungs but no

38、t detected in blood, not detected in blood, bone marrow, or spleen. bone marrow, or spleen. One year after PMT of CD45.1 + WT BMDMs into CD45.2+ One year after PMT of CD45.1 + WT BMDMs into CD45.2+ KO mice,flow cytometric detection of CD45.1 + cells KO mice,flow cytometric detection of CD45.1 + cell

39、s confirmed thease findings (Extended Data Fig.4e-g). confirmed thease findings (Extended Data Fig.4e-g). the percentage of CD45.1 +cells in the gated regions the percentage of CD45.1 +cells in the gated regions are shown are shown Results show that the transplanted Results show that the transplante

40、d macrophages remained in the lungs, macrophages remained in the lungs, primarily within the intra-alveolar space.primarily within the intra-alveolar space.The effects of the lung milieu on the phenotype of transplanted macrophages were evaluated by measuring cell-surface markers. One year after PMT

41、 of WT Lys-MGFP BMDMs into KO mice,similar to the phenotype of WT alveolar macrophages and different from recipient KO mice微阵列分析Microarray analysisMicroarray analysis Unsupervised hierarchicalUnsupervised hierarchicalclustering dendrogramclustering dendrogram层析聚层析聚类分析类分析heat-map of selected GM-CSF-h

42、eat-map of selected GM-CSF-regulated genesregulated genesExpression of genes regulated by GM-CSF was reduced in Expression of genes regulated by GM-CSF was reduced in KOmice and restored by PMT (Fig. 4, Extended Data Fig. KOmice and restored by PMT (Fig. 4, Extended Data Fig. 5a).5a).(a) Expression

43、of (a) Expression of Spi1 Spi1 (PU.1) and (PU.1) and Pparg Pparg (PPAR were (PPAR were confirmed by qRT-PCR using independent samplesconfirmed by qRT-PCR using independent samples(b)Venn diagrams showing (b)Venn diagrams showing numbers of genes whosenumbers of genes whoseexpression was altered in e

44、xpression was altered in alveolar macrophages from KO alveolar macrophages from KO c o m p a r e d t o W T m i c e c o m p a r e d t o W T m i c e (WTKO)or PMT-tr eated (WTKO)or PMT-tr eated compared to untreated KO compared to untreated KO mice (KOKO+PMT).Only mice (KOKO+PMT).Only genes with statis

45、tically genes with statistically significant changes of at least significant changes of at least two-fold were marked as two-fold were marked as increased (up arrows) or increased (up arrows) or decreased (down arrows). decreased (down arrows). Of 776 genes for which Of 776 genes for which expressio

46、n was disrupted in expression was disrupted in KO mice, PMT normalized KO mice, PMT normalized expression of 600 including expression of 600 including 80% of genes up-regulated80% of genes up-regulated and and 76% of genes down-regulated76% of genes down-regulated in KO compared to WT micein KO comp

47、ared to WT mice(Extended Data Fig. 5b).(Extended Data Fig. 5b).(d) Heat maps showing differentially expressed genes (d) Heat maps showing differentially expressed genes including PPARregulated genes, glycophospholipid including PPARregulated genes, glycophospholipid metabolism, peroxisome function a

48、poptosis, cell cycle metabolism, peroxisome function apoptosis, cell cycle control, Genes with increased or decreased transcript control, Genes with increased or decreased transcript levels are shown by red and blue colors, respectively.levels are shown by red and blue colors, respectively.Efficacy

49、of gene therapy by PMTEfficacy of gene therapy by PMTSince PMT in humans would likely employ autologous, Since PMT in humans would likely employ autologous, gene-corrected HSPC-derived macrophages, we evaluated gene-corrected HSPC-derived macrophages, we evaluated PMT of KO macrophages derived from LSK cells after PMT of KO macrophages derived from LSK cells after lentiviral vector (LV)-mediated lentiviral vector (LV)-mediated Csf2rb Csf2rb cDNA expression cDNA expression (Fig. 5a).(Fig. 5a).只有WT,KO+GM-R-GFP中的CD131+ 表达CD68+都表达(c) Western blotting to detect GM-CSF r