过氧化物酶体增殖活化受体-γ对炎症，免疫抑制和肺气肿生成的影响
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摘要
过氧化物酶体增殖活化受体-γPeroxisomeProliferator-ActivatedReceptor-γγPPARγ可减少炎症因子的表达，是一个抗炎转录调节因子。本实验室前期研究结果发现，当溶酶体酸性脂肪酶（Lysosomalacidlipase,LAL）缺乏时，阻断PPARγ配体的合成可以导致肺部严重的炎症和肺气肿，表明PPARγ的失活对这些病变起重要作用。为了探明PPARγ在肺中所扮演的病理生理作用，我们建立了CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠模型，在mRNA和蛋白质水平上，都检测到四环素诱导产生的dnPPARγ-Flag融合蛋白在肺II型上皮细胞的表达。我们假设PPARγ在肺II型上皮细胞中起了重要作用，过量表达负显性PPARγ（dominantnegativePPARγdnPPARγ）可抑制内源性PPARγ在肺II型上皮细胞里的功能，从而调控肺的炎症产生及病变的形成。主要结论如下：
①dnPPARγ在肺部的过量表达导致了肺气肿的形成。为了研究dnPPARγ在肺型上皮细胞中的过量表达对转基因小鼠肺部的影响，我们对4月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ转基因小鼠的肺部进行了组织学分析，我们观察到了在四环素诱导下的双转基因小鼠中肺气肿的形成。显微镜下观察HE染色的肺组织切片，用MetaMorph图像分析软件进行定量组织形态学分析。四环素诱导的双转基因小鼠的肺泡数量（65±9）显著低于那些没有被四环素诱导的转基因小鼠的肺泡数量（122±13）。在四环素诱导的双转基因小鼠中，平均线长度（Lm），肺泡面积，肺泡表面积和肺泡体积都较没有诱导的小鼠明显增加，表明dnPPARγ在肺部的过量表达导致了肺气肿的形成。PPARγ在肺组织中可负调节的基因还有金属基质蛋白酶（Matrixmetalloproteinase,MMPs）。MMPs的上调可降解细胞外基质，使肺泡间隙结构弱化，从而导致肺气肿。对4月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠进行研究，在四环素诱导的双转基因小鼠中，MMP2,MMP7,MMP8,MMP9,MMP12在整个肺部和肺II型上皮细胞中的mRNA表达水平显著高于那些没有四环素诱导的双转基因小鼠。在被四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠的肺腔灌洗液（Bronchioalveolarlarvagefluid,BALF）中，MMP12和MMP9也比没有四环素诱导的BALF中的酶活性增强了，这有助于肺气肿的形成。因此，PPARγ的失活导致的MMPs的合成和分泌对肺气肿的形成起到了一定的作用。
②炎症细胞因子的上调表达。PPARγ是一个抗炎分子，dnPPARγ的过量表达抑制了内源PPARγ的抗炎活性，我们研究了CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠中的炎症因子的表达。dnPPARγ在四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠中的过量表达可通过竞争性结合于炎症因子基因启动子上的PPARγE位点，取代PPARγ与启动子的结合，从而在转录水平上抑制了内源PPARγ的抗炎活性，使炎症因子的表达上调。为了验证这一假设，我们采用染色质免疫共沉淀(ChromatinImmunoprecipitation，ChIP)的方法分析了月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺中dnPPARγ和Api6,IL-1,IL-6,MMP12和TNF-基因启动子上PPARγE位点的结合情况。抗flag多克隆抗体被用于免疫沉淀dnPPARγ-flag/DNA复合物。ChIP的实验结果显示，在四环素诱导的双转基因小鼠中，沉淀下来的启动子DNA片段的信号要高于那些没有被四环素诱导的小鼠，表明dnPPARγ取代内源PPARγ与启动子的结合，抑制了其抗炎活性。为了研究dnPPARγ的过量表达是否可以上调双转基因小鼠体内炎症因子的表达水平，我们提取月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺总细胞和肺II型上皮细胞的mR，用Real-timePCR的方法定量检测了多个炎症细胞因子的表达水平。与没有四环素诱导的小鼠相比，受四环素诱导的双转基因小鼠的肺总细胞和肺II型上皮细胞中的IFN-,IFN-,IL-1,IL-6,IL-13,TNF-的表达水平升高。用ELISA检测了IL-1,IL-6,TNF-在BALF中的蛋白质浓度，与没有四环素诱导的小鼠相比，受四环素诱导的双转基因小鼠的BALF中IL-1,IL-6,TNF-的蛋白质浓度的的也升高。
③髓源性抑制细胞向肺部的侵入。为了研究dnPPARγ的过量表达导致的炎症细胞因子的上调是否会导致炎症细胞的向肺的入侵，提取1，4，8月龄的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠的外周血细胞和肺细胞，用CD11b，GR-1抗体染色，流式细胞仪分析小鼠的炎症细胞。与没有四环素诱导的小鼠相比，受四环素诱导的双转基因小鼠肺和血液细胞中的CD11b~+GR-1~+细胞在1，4，8月时间点都逐步上升。HE染色显示大量的炎症细胞在受四环素诱导的双转基因小鼠肺部聚集，Mac2免疫组织化学染色结果表明，这些炎症细胞是髓源性细胞。Kwik-Diff染色结果也显示在受四环素诱导的双转基因小鼠BALF中，炎症细胞大量增加。该研究结果表明，由于dnPPARγ在肺II型上皮细胞的过量表达，使髓源性细胞处于非成熟阶段，其分化被阻断，不能分化成为成熟的巨噬细胞和粒细胞。这种非成熟的CD11b~+GR-1~+细胞即髓源性抑制细胞（Myeloid-derivedsuppressorcells，MDSCs），它可抑制T细胞的增殖和功能，搅乱免疫监视，阻止免疫系统功能。我们的研究结果也表明，在四环素素诱导4个月的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠外周血与肺中，伴随着CD11b+GR-1+MDSCs细胞的增多，CD3~+，CD4~+和CD8~+T细胞的数量减少。
④细胞内信号分子的表达。在受四环素诱导的肺II型上皮细胞中，由于dnPPARγ的过量表达，Stat1，Stat2，Stat3，Stat4，Erk1/2和P38等一系列细胞内信号分子的磷酸化增多，这可能是由于炎症细胞因子刺激物通过自分泌途径上调了这些信号分子的表达。这些研究结果表明，PPARγ信号通路的失活不仅引起炎症细胞因子的上调，同时激活了一系列的肺II型上皮细胞中细胞内信号分子路径。同时，与没有四环素诱导的小鼠相比，在受四环素诱导的dnPPARγ的过量表达的转基因小鼠肺和血液的CD11b~+GR-1~+MDSCs细胞中，pStat3，pErk和pP38与其总蛋白的比值增高。综上结果表明，dnPPARγ在CCSP-rtTA/(tetO)_7-dnPPARγ双转基因小鼠肺II型上皮细胞中的过量表达，诱导了炎症分子的产生，通过自分泌机制使肺II型上皮细胞内的癌症信号分子通路激活，并通过旁分泌机制使CD11b~+GR-1~+MDSCs内的癌症信号分子通路激活。
⑤髓源性抑制细胞MDSCs对T细胞增殖，活性和功能影响的体外研究。为了研究CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠的MDSCs对T细胞的影响，CFSE标记的CD4~+T细胞，用抗CD3单克隆抗体和抗CD28单克隆抗体刺激T细胞的增殖，与MDSCs共培养4天，流式细胞仪检测T细胞的增殖情况。实验结果表明，来源于四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞对野生型WT脾脏CD4~+T细胞增殖具有抑制作用。同样，来源于四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞对野生型WT脾脏CD4~+T细胞的活性具有抑制作用，其表达CD69的量减少。用ELISA的方法检测共培养体系中的IL-2, IL-4, IFN-的蛋白浓度来研究MDSCs对CD4~+T细胞功能的影响。实验结果表明，来源于四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞与野生型WT脾脏CD4~+T细胞共培养后，其表达IL-2, IL-4, IFN-的量减少。用AnnexinV标记CD4~+T细胞来检测MDSCs对T细胞凋亡的影响。实验结果表明，CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞与WT脾脏CD4~+T细胞共培养时，与来源于没有四环素诱导的相比，来源于四环素诱导的双转基因小鼠肺的CD11b~+GR-1~+细胞使野生型WT脾脏CD4~+T细胞的凋亡增加。
综上所述，dnPPARγ的过量表达阻断了内源性PPARγ对炎症基因的抑制作用，包括细胞因子和MMPs。这促使了炎症细胞，特别是MDSCs向肺的侵润，改变了原有的微环境。在非免疫应答中，肺II型上皮细胞中多个胞内信号分子的活化导致肺的改变，从而有助于肺气肿的形成。这些研究结果表明，LAL/配体/PPARγ路径在抑制肺部炎症，维持肺泡正常结构中扮演重要的作用。
Peroxisome Proliferator-Activated Receptor-γγ PPARγ is an anti-inflammatorytranscriptional factor by reducing pro-inflammatory cytokine expression. Previously, wereported that blockage of PPARγ ligand synthesis during LAL deficiency causedexuberant inflammation and emphysema in the lung, implicating that PPARγ inactivation is responsible for these phenotypes. In order to identify thephathophysiological roles of PPARγ in the lung, a doxycycline-controlledCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mouse model was generated tooverexpress dnPPARγ in lung epithelial cells to block the endogenous function ofPPARγ. In this bitransgenic model, doxycycline treatment induced dnPPARγ-Flagfusion protein expression in AT II epithelial cells at both mRNA and protein levels.Flow cytometry analysis showed dnPPARγ-Flag fusion protein expression associatedwith AT II epithelial cells. We hypothesize that PPARγ in AT II epithelial cells playsimportant roles in controlling pulmonary inflammation and associated pathogenesis.The main results are as follows:
①To assess pathogenic phenotypes in the lung as a result of dnPPARγ overexpression in AT II epithelial cells, CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice with4-month doxycycline treatment were investigated by histologicalanalysis.Histopathological analysis revealed emphysema in the lung ofdoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. Clusteredinflammatory cell accumulation was observed in some lung areas. Quantitative analysisby the MetaMorph imaging software showed that alveolar numbers ofdoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ mice (65±9) were much lessthan those of untreated bitransgenic mice (122±13). The mean cord length (Lm),alveolar area, alveolar sphere surface area and alveolar volume were all significantlyincreased in doxycycline-treated bitransgenic mice compared with those of untreatedbitransgenic mice. After dnPPARγ overexpression, the mRNA levels of MMP12, alongwith MMP2, MMP7, MMP8and MMP9were all up-regulated in AT II epithelial cellsfrom doxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. Theenzymatic activity of MMP9and MMP12were increased in BALF ofdoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. Thus, MMP synthesis and secretion were induced by inactivation of PPARγ in AT II epithelialcells that contributed to emphysema formation.
②Since PPARγ is an anti-inflammatory molecule, it is important to evaluatewhether the pro-inflammatory molecules were up-regulated inCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. These molecules are potentiallyimportant for triggering and recruiting immune cells into the lung to initiateinflammation and emphysema. PPARγ, which binds to the specific DNA sites (PPARγ E)on the promoters after ligand binding, prevents transcriptional events ofpro-inflammatory molecules. ChIP assay was performed to investigate the associationbetween dnPPARγ and PPARγ E sites within the Api6, IL-1, IL-6, MMP12and TNF-promoters in2-month doxycycline-treated or untreatedCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic lung. The putative PPARγ E siteswithin the promoters of Api6, IL-1, IL-6, MMP12and TNF-were identified.Anti-Flag polyclonal antibody was used to immuno-precipitate the dnPPARγ-Flag/DNAcomplex. In doxycycline-treated mice, the signals of precipitated promoter DNA werestronger than those of untreated controls by quantitative Real-Time PCR analysis. Theresults showed that dnPPARγ-Flag fusion protein was able to bind to the promoters ofthese pro-inflammatory genes. To determine if dnPPARγ overexpression indeedup-regulates proinflammatory cytokines in vivo, total RNAs were purified from thewhole lung and AT II epithelial cells of2-month doxycycline-treated or untreatedCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. The expression levels ofmultiple cytokines were quantitatively determined by Real-Time PCR. Among them,mRNA expression levels of IFN-, IFN-, IL-1, IL-6, IL-13, and TNF-indoxycycline-treated mice were increased compared with those in doxycycline-untreatedmice. In BALF, protein concentrations of secreted IL-1, IL-6and TNF-were steadilyincreased as measured by ELISA compared with those in untreated mice.
③Up-regulation of pro-inflammatory cytokines suggests that overexpression ofdnPPARγ may induce inflammation that contributes to emphysema. To identifywhether overexpression of dnPPARγ induces inflammatory cell infiltration in the lungof CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice as a result ofpro-inflammatory cytokine up-regulation, peripheral blood mononuclear cells (PBMCs)and lung mononuclear cells were isolated from1,4,8-month doxycycline-treated oruntreated mice and stained with fluorochrome-conjugated anti-mouse CD11b (formonocytes) and Gr-1(for neutrophils) antibodies for FACS analysis. Compared with doxycycline-untreated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice,immature CD11b~+GR-1~+MDSCs were steadily increased in the blood and lung of4,8-month doxycycline-treated mice. Kwik-Diff staining showed significant increase ofinflammatory cells in the BALF of doxycycline-treated mice. This suggests thatmyeloid cells were blocked at the immature stage to differentiate into maturemacrophages and neutrophils after dnPPARγ overexpression in AT II epithelial cells. Itis known that MDSCs suppress T cell proliferation and function to subvert immunesurveillance and prevent the immune system from eliminating unwanted pathogenicevents. Indeed, decreased numbers of CD3~+, CD4~+and CD8~+T cells were observed indoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic lungs incorrelation with CD11b~+Gr-1~+MDSCs expansion4months after doxycycline treatment.In these assays, the absolute numbers of CD3, CD4and CD8T cells were also reduced.
④In AT II epithelial cells, a set of intracelluar signaling molecules, includingStat1, Stat2, Stat3, Stat4, Erk1/2, NF B, p38and PU.1, showed increasedphosphorylation (activation) by dnPPARγ overexpression. This can be a result ofpro-inflammatory cytokine stimuli up-regulation through autocrine pathways. Theseresults indicate that inactivation of the PPARγ pathway not only up-regulatedpro-inflammatory cytokines, but also activated unique sets of intracellular signalingmolecules in AT II epithelial cells. In CD11b~+Gr-1~+MDSCs from dnPPARγ overexpressed bitransgenic mice, activation of Stat3, Erk1/2, NF B, p38and Akt wereobserved in doxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic micecompared with those of untreated bitransgenic mice. It appears that inflammatorymolecules induced by dnPPARγ overexpression in the milieu of doxycycline-treatedCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic lung activated oncogenic intracellularsignaling molecules in AT II epithelial cells through the autocrine mechanism and thosein CD11b~+Gr-1~+MDSCs through the paracrine mechanism.
⑤To measure the effect of CD11b~+Gr-1~+cells on T cell proliferation inCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice, CFSE labeled wild type splenicCD4~+T cells were stimulated with anti-CD3and anti-CD28mAb in the presence ofCD11b~+Gr-1~+cells that were isolated from the bone marrow, spleen and lung ofdoxycycline-treated or untreated mice. After4days co-culturing, T cell proliferationwas measured by CFSE labeling dilution and flow cytometry. As a result, TCRstimulated T cell proliferation (represented by multiple peaks) was suppressed byCD11b~+Gr-1~+cells from the lung of doxycycline-treated bitransgenic mice compared with CD11b~+Gr-1~+cells of untreated bitransgenic mice. Therefore, the defect caused bydnPPARγ overexpression in AT II epithelial cells was restricted to the lung and blood.A similar observation was made in the CD69(a T cell activation marker) expressionassay. After48hours co-culturing, CD11b~+Gr-1~+cells from the lung ofdoxycycline-treated bitransgenic mice suppressed anti-CD3Ab plus anti-CD28Ab-stimulated CD69expression compared with CD11b~+Gr-1~+cells of untreatedbitransgenic mice. In functional analysis, secretion of lymphokines was measured inMDSCs/CD4T cell co-culturing medium by ELISA. CD11b~+Gr-1~+cells from the lungsof doxycycline-treated mice showed reduced secretion of IL-2, IL-4and IFN-afterTCR activation compared with CD11b~+Gr-1~+cells of untreated bitransgenic mice. Todetermine whether CD4~+T cell reduction is associated with the apoptosis, CD4~+T cellswere analyzed by Annexin V staining. Only CD4~+T cells that were co-cultured withCD11b~+Gr-1~+cells from the lungs of doxycycline-treated bitransgenic mice showed theincreased apoptotic activity compared with CD11b~+Gr-1~+cells of untreated bitransgenicmice. Thus, dnPPARγ-induced myeloid defect had a profound impact on T cellproliferation and function.
In summary, overexpression of dnPPARγ relieved endogenous PPARγ suppressionon the transcriptional activities of pro-inflammatory genes including cytokines andMMPs. This action promoted inflammatory cell infiltration (especially MDSCs) into thelung and changed the regional microenvironment. In the non-immune response, AT IIepithelial cells were changed as evident by activation of multiple intracellular signalingmolecules. As a consequence, emphysema was formed. These observations support aconcept that the LAL/ligands/PPARγ axis plays a critical role in maintaining alveolarhomeostasis by suppressing pulmonary inflammation.
①dnPPARγ在肺部的过量表达导致了肺气肿的形成。为了研究dnPPARγ在肺型上皮细胞中的过量表达对转基因小鼠肺部的影响，我们对4月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ转基因小鼠的肺部进行了组织学分析，我们观察到了在四环素诱导下的双转基因小鼠中肺气肿的形成。显微镜下观察HE染色的肺组织切片，用MetaMorph图像分析软件进行定量组织形态学分析。四环素诱导的双转基因小鼠的肺泡数量（65±9）显著低于那些没有被四环素诱导的转基因小鼠的肺泡数量（122±13）。在四环素诱导的双转基因小鼠中，平均线长度（Lm），肺泡面积，肺泡表面积和肺泡体积都较没有诱导的小鼠明显增加，表明dnPPARγ在肺部的过量表达导致了肺气肿的形成。PPARγ在肺组织中可负调节的基因还有金属基质蛋白酶（Matrixmetalloproteinase,MMPs）。MMPs的上调可降解细胞外基质，使肺泡间隙结构弱化，从而导致肺气肿。对4月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠进行研究，在四环素诱导的双转基因小鼠中，MMP2,MMP7,MMP8,MMP9,MMP12在整个肺部和肺II型上皮细胞中的mRNA表达水平显著高于那些没有四环素诱导的双转基因小鼠。在被四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠的肺腔灌洗液（Bronchioalveolarlarvagefluid,BALF）中，MMP12和MMP9也比没有四环素诱导的BALF中的酶活性增强了，这有助于肺气肿的形成。因此，PPARγ的失活导致的MMPs的合成和分泌对肺气肿的形成起到了一定的作用。
②炎症细胞因子的上调表达。PPARγ是一个抗炎分子，dnPPARγ的过量表达抑制了内源PPARγ的抗炎活性，我们研究了CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠中的炎症因子的表达。dnPPARγ在四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠中的过量表达可通过竞争性结合于炎症因子基因启动子上的PPARγE位点，取代PPARγ与启动子的结合，从而在转录水平上抑制了内源PPARγ的抗炎活性，使炎症因子的表达上调。为了验证这一假设，我们采用染色质免疫共沉淀(ChromatinImmunoprecipitation，ChIP)的方法分析了月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺中dnPPARγ和Api6,IL-1,IL-6,MMP12和TNF-基因启动子上PPARγE位点的结合情况。抗flag多克隆抗体被用于免疫沉淀dnPPARγ-flag/DNA复合物。ChIP的实验结果显示，在四环素诱导的双转基因小鼠中，沉淀下来的启动子DNA片段的信号要高于那些没有被四环素诱导的小鼠，表明dnPPARγ取代内源PPARγ与启动子的结合，抑制了其抗炎活性。为了研究dnPPARγ的过量表达是否可以上调双转基因小鼠体内炎症因子的表达水平，我们提取月龄CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺总细胞和肺II型上皮细胞的mR，用Real-timePCR的方法定量检测了多个炎症细胞因子的表达水平。与没有四环素诱导的小鼠相比，受四环素诱导的双转基因小鼠的肺总细胞和肺II型上皮细胞中的IFN-,IFN-,IL-1,IL-6,IL-13,TNF-的表达水平升高。用ELISA检测了IL-1,IL-6,TNF-在BALF中的蛋白质浓度，与没有四环素诱导的小鼠相比，受四环素诱导的双转基因小鼠的BALF中IL-1,IL-6,TNF-的蛋白质浓度的的也升高。
③髓源性抑制细胞向肺部的侵入。为了研究dnPPARγ的过量表达导致的炎症细胞因子的上调是否会导致炎症细胞的向肺的入侵，提取1，4，8月龄的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠的外周血细胞和肺细胞，用CD11b，GR-1抗体染色，流式细胞仪分析小鼠的炎症细胞。与没有四环素诱导的小鼠相比，受四环素诱导的双转基因小鼠肺和血液细胞中的CD11b~+GR-1~+细胞在1，4，8月时间点都逐步上升。HE染色显示大量的炎症细胞在受四环素诱导的双转基因小鼠肺部聚集，Mac2免疫组织化学染色结果表明，这些炎症细胞是髓源性细胞。Kwik-Diff染色结果也显示在受四环素诱导的双转基因小鼠BALF中，炎症细胞大量增加。该研究结果表明，由于dnPPARγ在肺II型上皮细胞的过量表达，使髓源性细胞处于非成熟阶段，其分化被阻断，不能分化成为成熟的巨噬细胞和粒细胞。这种非成熟的CD11b~+GR-1~+细胞即髓源性抑制细胞（Myeloid-derivedsuppressorcells，MDSCs），它可抑制T细胞的增殖和功能，搅乱免疫监视，阻止免疫系统功能。我们的研究结果也表明，在四环素素诱导4个月的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠外周血与肺中，伴随着CD11b+GR-1+MDSCs细胞的增多，CD3~+，CD4~+和CD8~+T细胞的数量减少。
④细胞内信号分子的表达。在受四环素诱导的肺II型上皮细胞中，由于dnPPARγ的过量表达，Stat1，Stat2，Stat3，Stat4，Erk1/2和P38等一系列细胞内信号分子的磷酸化增多，这可能是由于炎症细胞因子刺激物通过自分泌途径上调了这些信号分子的表达。这些研究结果表明，PPARγ信号通路的失活不仅引起炎症细胞因子的上调，同时激活了一系列的肺II型上皮细胞中细胞内信号分子路径。同时，与没有四环素诱导的小鼠相比，在受四环素诱导的dnPPARγ的过量表达的转基因小鼠肺和血液的CD11b~+GR-1~+MDSCs细胞中，pStat3，pErk和pP38与其总蛋白的比值增高。综上结果表明，dnPPARγ在CCSP-rtTA/(tetO)_7-dnPPARγ双转基因小鼠肺II型上皮细胞中的过量表达，诱导了炎症分子的产生，通过自分泌机制使肺II型上皮细胞内的癌症信号分子通路激活，并通过旁分泌机制使CD11b~+GR-1~+MDSCs内的癌症信号分子通路激活。
⑤髓源性抑制细胞MDSCs对T细胞增殖，活性和功能影响的体外研究。为了研究CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠的MDSCs对T细胞的影响，CFSE标记的CD4~+T细胞，用抗CD3单克隆抗体和抗CD28单克隆抗体刺激T细胞的增殖，与MDSCs共培养4天，流式细胞仪检测T细胞的增殖情况。实验结果表明，来源于四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞对野生型WT脾脏CD4~+T细胞增殖具有抑制作用。同样，来源于四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞对野生型WT脾脏CD4~+T细胞的活性具有抑制作用，其表达CD69的量减少。用ELISA的方法检测共培养体系中的IL-2, IL-4, IFN-的蛋白浓度来研究MDSCs对CD4~+T细胞功能的影响。实验结果表明，来源于四环素诱导的CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞与野生型WT脾脏CD4~+T细胞共培养后，其表达IL-2, IL-4, IFN-的量减少。用AnnexinV标记CD4~+T细胞来检测MDSCs对T细胞凋亡的影响。实验结果表明，CCSP-rtTA/(tetO)_7-CMV-dnPPARγ双转基因小鼠肺的CD11b~+GR-1~+细胞与WT脾脏CD4~+T细胞共培养时，与来源于没有四环素诱导的相比，来源于四环素诱导的双转基因小鼠肺的CD11b~+GR-1~+细胞使野生型WT脾脏CD4~+T细胞的凋亡增加。
综上所述，dnPPARγ的过量表达阻断了内源性PPARγ对炎症基因的抑制作用，包括细胞因子和MMPs。这促使了炎症细胞，特别是MDSCs向肺的侵润，改变了原有的微环境。在非免疫应答中，肺II型上皮细胞中多个胞内信号分子的活化导致肺的改变，从而有助于肺气肿的形成。这些研究结果表明，LAL/配体/PPARγ路径在抑制肺部炎症，维持肺泡正常结构中扮演重要的作用。
Peroxisome Proliferator-Activated Receptor-γγ PPARγ is an anti-inflammatorytranscriptional factor by reducing pro-inflammatory cytokine expression. Previously, wereported that blockage of PPARγ ligand synthesis during LAL deficiency causedexuberant inflammation and emphysema in the lung, implicating that PPARγ inactivation is responsible for these phenotypes. In order to identify thephathophysiological roles of PPARγ in the lung, a doxycycline-controlledCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mouse model was generated tooverexpress dnPPARγ in lung epithelial cells to block the endogenous function ofPPARγ. In this bitransgenic model, doxycycline treatment induced dnPPARγ-Flagfusion protein expression in AT II epithelial cells at both mRNA and protein levels.Flow cytometry analysis showed dnPPARγ-Flag fusion protein expression associatedwith AT II epithelial cells. We hypothesize that PPARγ in AT II epithelial cells playsimportant roles in controlling pulmonary inflammation and associated pathogenesis.The main results are as follows:
①To assess pathogenic phenotypes in the lung as a result of dnPPARγ overexpression in AT II epithelial cells, CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice with4-month doxycycline treatment were investigated by histologicalanalysis.Histopathological analysis revealed emphysema in the lung ofdoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. Clusteredinflammatory cell accumulation was observed in some lung areas. Quantitative analysisby the MetaMorph imaging software showed that alveolar numbers ofdoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ mice (65±9) were much lessthan those of untreated bitransgenic mice (122±13). The mean cord length (Lm),alveolar area, alveolar sphere surface area and alveolar volume were all significantlyincreased in doxycycline-treated bitransgenic mice compared with those of untreatedbitransgenic mice. After dnPPARγ overexpression, the mRNA levels of MMP12, alongwith MMP2, MMP7, MMP8and MMP9were all up-regulated in AT II epithelial cellsfrom doxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. Theenzymatic activity of MMP9and MMP12were increased in BALF ofdoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. Thus, MMP synthesis and secretion were induced by inactivation of PPARγ in AT II epithelialcells that contributed to emphysema formation.
②Since PPARγ is an anti-inflammatory molecule, it is important to evaluatewhether the pro-inflammatory molecules were up-regulated inCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. These molecules are potentiallyimportant for triggering and recruiting immune cells into the lung to initiateinflammation and emphysema. PPARγ, which binds to the specific DNA sites (PPARγ E)on the promoters after ligand binding, prevents transcriptional events ofpro-inflammatory molecules. ChIP assay was performed to investigate the associationbetween dnPPARγ and PPARγ E sites within the Api6, IL-1, IL-6, MMP12and TNF-promoters in2-month doxycycline-treated or untreatedCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic lung. The putative PPARγ E siteswithin the promoters of Api6, IL-1, IL-6, MMP12and TNF-were identified.Anti-Flag polyclonal antibody was used to immuno-precipitate the dnPPARγ-Flag/DNAcomplex. In doxycycline-treated mice, the signals of precipitated promoter DNA werestronger than those of untreated controls by quantitative Real-Time PCR analysis. Theresults showed that dnPPARγ-Flag fusion protein was able to bind to the promoters ofthese pro-inflammatory genes. To determine if dnPPARγ overexpression indeedup-regulates proinflammatory cytokines in vivo, total RNAs were purified from thewhole lung and AT II epithelial cells of2-month doxycycline-treated or untreatedCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice. The expression levels ofmultiple cytokines were quantitatively determined by Real-Time PCR. Among them,mRNA expression levels of IFN-, IFN-, IL-1, IL-6, IL-13, and TNF-indoxycycline-treated mice were increased compared with those in doxycycline-untreatedmice. In BALF, protein concentrations of secreted IL-1, IL-6and TNF-were steadilyincreased as measured by ELISA compared with those in untreated mice.
③Up-regulation of pro-inflammatory cytokines suggests that overexpression ofdnPPARγ may induce inflammation that contributes to emphysema. To identifywhether overexpression of dnPPARγ induces inflammatory cell infiltration in the lungof CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice as a result ofpro-inflammatory cytokine up-regulation, peripheral blood mononuclear cells (PBMCs)and lung mononuclear cells were isolated from1,4,8-month doxycycline-treated oruntreated mice and stained with fluorochrome-conjugated anti-mouse CD11b (formonocytes) and Gr-1(for neutrophils) antibodies for FACS analysis. Compared with doxycycline-untreated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice,immature CD11b~+GR-1~+MDSCs were steadily increased in the blood and lung of4,8-month doxycycline-treated mice. Kwik-Diff staining showed significant increase ofinflammatory cells in the BALF of doxycycline-treated mice. This suggests thatmyeloid cells were blocked at the immature stage to differentiate into maturemacrophages and neutrophils after dnPPARγ overexpression in AT II epithelial cells. Itis known that MDSCs suppress T cell proliferation and function to subvert immunesurveillance and prevent the immune system from eliminating unwanted pathogenicevents. Indeed, decreased numbers of CD3~+, CD4~+and CD8~+T cells were observed indoxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic lungs incorrelation with CD11b~+Gr-1~+MDSCs expansion4months after doxycycline treatment.In these assays, the absolute numbers of CD3, CD4and CD8T cells were also reduced.
④In AT II epithelial cells, a set of intracelluar signaling molecules, includingStat1, Stat2, Stat3, Stat4, Erk1/2, NF B, p38and PU.1, showed increasedphosphorylation (activation) by dnPPARγ overexpression. This can be a result ofpro-inflammatory cytokine stimuli up-regulation through autocrine pathways. Theseresults indicate that inactivation of the PPARγ pathway not only up-regulatedpro-inflammatory cytokines, but also activated unique sets of intracellular signalingmolecules in AT II epithelial cells. In CD11b~+Gr-1~+MDSCs from dnPPARγ overexpressed bitransgenic mice, activation of Stat3, Erk1/2, NF B, p38and Akt wereobserved in doxycycline-treated CCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic micecompared with those of untreated bitransgenic mice. It appears that inflammatorymolecules induced by dnPPARγ overexpression in the milieu of doxycycline-treatedCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic lung activated oncogenic intracellularsignaling molecules in AT II epithelial cells through the autocrine mechanism and thosein CD11b~+Gr-1~+MDSCs through the paracrine mechanism.
⑤To measure the effect of CD11b~+Gr-1~+cells on T cell proliferation inCCSP-rtTA/(tetO)_7-CMV-dnPPARγ bitransgenic mice, CFSE labeled wild type splenicCD4~+T cells were stimulated with anti-CD3and anti-CD28mAb in the presence ofCD11b~+Gr-1~+cells that were isolated from the bone marrow, spleen and lung ofdoxycycline-treated or untreated mice. After4days co-culturing, T cell proliferationwas measured by CFSE labeling dilution and flow cytometry. As a result, TCRstimulated T cell proliferation (represented by multiple peaks) was suppressed byCD11b~+Gr-1~+cells from the lung of doxycycline-treated bitransgenic mice compared with CD11b~+Gr-1~+cells of untreated bitransgenic mice. Therefore, the defect caused bydnPPARγ overexpression in AT II epithelial cells was restricted to the lung and blood.A similar observation was made in the CD69(a T cell activation marker) expressionassay. After48hours co-culturing, CD11b~+Gr-1~+cells from the lung ofdoxycycline-treated bitransgenic mice suppressed anti-CD3Ab plus anti-CD28Ab-stimulated CD69expression compared with CD11b~+Gr-1~+cells of untreatedbitransgenic mice. In functional analysis, secretion of lymphokines was measured inMDSCs/CD4T cell co-culturing medium by ELISA. CD11b~+Gr-1~+cells from the lungsof doxycycline-treated mice showed reduced secretion of IL-2, IL-4and IFN-afterTCR activation compared with CD11b~+Gr-1~+cells of untreated bitransgenic mice. Todetermine whether CD4~+T cell reduction is associated with the apoptosis, CD4~+T cellswere analyzed by Annexin V staining. Only CD4~+T cells that were co-cultured withCD11b~+Gr-1~+cells from the lungs of doxycycline-treated bitransgenic mice showed theincreased apoptotic activity compared with CD11b~+Gr-1~+cells of untreated bitransgenicmice. Thus, dnPPARγ-induced myeloid defect had a profound impact on T cellproliferation and function.
In summary, overexpression of dnPPARγ relieved endogenous PPARγ suppressionon the transcriptional activities of pro-inflammatory genes including cytokines andMMPs. This action promoted inflammatory cell infiltration (especially MDSCs) into thelung and changed the regional microenvironment. In the non-immune response, AT IIepithelial cells were changed as evident by activation of multiple intracellular signalingmolecules. As a consequence, emphysema was formed. These observations support aconcept that the LAL/ligands/PPARγ axis plays a critical role in maintaining alveolarhomeostasis by suppressing pulmonary inflammation.
引文
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