Influence Of Peroxisome Proliferator-Activated Receptor-γ To Inflammation, Immunosuppression And Emphysema

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.