Alveolar Macrophage Polarization And Its Mechanism In Carbon Nanotube-induced Pulmonary Fibrosis

BackgroundWith substantial progress of nanotechnology,carbon nanotubes（CNTs）are widely used in a variety of industrial and commercial applications due to their unique physicochemical properties.CNTs are classified to single-walled CNT（SWCNT）and multi-walled CNT（MWCNT）.With rapid expansion of CNTs production and application,the risk of human exposure to CNTs substantially raises,such as lung function injury,lung inflammation and pulmonary fibrosis.It has been one of the urgent public health problems that how to prevent the potential harm of CNTs exposure to the human.Objective1.The role of AMs polarization in CNTs-induced pulmonary fibrosis in mice models.2.The mechanism that CNTs promote AMs toward M2 subtype-mediated epithelial-mesenchymal transition（EMT）in bronchial epithelial cells and fibroblast-myofibroblast transition（FMT）in primary lung fibroblasts in vitro.3.The molecular mechanism of SWCNT-upregulated M2 AMs polarization using in vivo and vitro（si RNA interference）experiments.Methods1.The characterization of CNTs:The morphology and structure were observed by high-resolution transmission electron microscopy.Zeta-potential was performed to observe the stability of the CNTs solution.Sedimentation kinetics were determined among three types of CNTs by sedimentation experiments.Endotoxin levels of CNTs were detected in sterilized solutions using Endotoxin Quant Kit.2.Vitro experiments2.1.To observe the effects of three types of CNTs on AMs polarization at different time points in vitro,the primary AM was obtained from C57BL/6J mice（8-10 week）,and AMs were divided into Control group,M1/M2 polarization group,SWCNT group,MWCNT（s）group and MWCNT（l）group.AMs in Control group,M1 polarization group and M2 polarization group were treated with PBS,LPS（48 h）+IFN-γ（24 h）（M1polarization condition）and IL-4（M2 polarization condition）for 48 h,respectively.AMs in SWCNT group,MWCNT（s）group and MWCNT（l）group were separately exposed to50μg/ml SWCNT,short-type MWCNT and long-type MWCNT for 6 h,12 h and 24 h under M1/M2 polarization conditions,then the level of AMs polarization was evaluated.2.2.To observe the effects of three types of CNTs-exposed AMs on EMT in bronchial epithelial cells and FMT in primary lung fibroblasts,the supernatants in CNTs-exposed AMs described in 2.1 section were collected as conditioned medium（CM）to culture BEAS-2B cells and primary lung fibroblasts.The cells were collected at 48 h after CM culture.Protein expressions of E-cad and Vimentin in BEAS-2B cells andα-SMA and CollagenⅠin primary lung fibroblasts were detected,then EMT and FMT were evaluated.2.3 To explore the mechanism that CNTs promote AMs toward M2 subtype-mediated EMT in bronchial epithelial cells and FMT in primary lung fibroblasts,TGF-β,IL-10and TGF-βinhibitor were used to treat BEAS-2B cells and primary lung fibroblasts based on CM experiments.Protein expressions of E-cad and Vimentin in BEAS-2B cells andα-SMA in primary lung fibroblasts were detected,then EMT and FMT were evaluated.2.4.To explore whether C/EBPβregulates M2 AMs polarization-mediated EMT in bronchial epithelial cells and FMT in primary lung fibroblasts,the primary AM was obtained from C57BL/6J mice,and AMs were divided into Control group,NC（normal control）group,M2 polarization group,SWCNT group,SWCNT+NC group and SWCNT+C/EBPβsi RNA group.The treatment of AMs in Control group,M2polarization group and SWCNT group were described in 2.1 section,AMs in NC group were treated with NC si RNA,as well as AMs in SWCNT+NC group and SWCNT+C/EBPβsi RNA group were treated with NC si RNA and C/EBPβsi RNA based on SWCNT exposure.Then,the supernatant of culture medium in each group were collected as CM,which were used to culture BEAS-2B cells and primary lung fibroblasts for 48 h.Protein expressions of E-cad and Vimentin in BEAS-2B cells andα-SMA and CollagenⅠin primary lung fibroblasts were detected,then EMT and FMT were evaluated.3.Vivo experiment3.1.To observe the change of AMs polarization and the extent of pulmonary fibrosis during the whole stage from acute inflammation to chronic fibrosis induced by three types of CNTs,C57BL/6J mice（8-10 week）were divided into Control group,SWCNT group,MWCNT（s）group and MWCNT（l）group（12 mice each group）.Mice were directly intratracheally instilled with 40μg SWCNT（SWCNT group）,short-type MWCNT（MWCNT（s）group）and long-type MWCNT（MWCNT（l）group）,respectively,while mice in CTRL group were administrated with PBS.Then,mice were sacrificed and the lung tissue and bronchoalveolar lavage fluid（BALF）were collected on days 3,7 and 28.AMs were isolated from BALF,then pulmonary fibrosis and AMs polarization were evaluated.3.2.SWCNT was selected for subsequent experiments to explore the potential mechanism in consideration that SWCNT showed stronger capacity to induce pulmonary fibrosis compared to MWCNT.To explore the role of M2 AMs polarization in SWCNT-induced pulmonary fibrosis,C57BL/6J mice（8-10 week）were divided into CTRL group,SWCNT group,SWCNT+Ig G group and SWCNT+anti IL-4 group（12mice each group）.Mice in SWCNT group,SWCNT+Ig G group and SWCNT+anti IL-4group were directly intratracheally instilled with 40μg SWCNT,while mice in CTRL group were administrated with PBS.Mice in SWCNT+Ig G group and SWCNT+anti IL-4 group were injected（ip）with 1 mg Ig G and IL-4 neutralizing antibody once a week.On day 28 after SWCNT administration,mice were sacrificed and the lung tissue and BALF were collected.AMs were isolated from BALF,then pulmonary fibrosis and AMs polarization were evaluated.3.3.To explore the possible mechanism of SWCNT-upregulated M2 AMs polarization,C57BL/6J mice（8-10 week）were divided into CTRL group and SWCNT group（12mice each group）.Mice in CTRL and SWCNT groups were directly intratracheally instilledd with PBS and 40μg SWCNT,respectively.On day 28 after SWCNT administration,mice were sacrificed and AMs were isolated from BALF.Then we detected m RNA expressions of 15 candidate genes-associated M2 macrophage polarization（AMPKα,BMP4,B7-H3,C/EBPβ,Fox O1,Herpub1,IRF4,KLF4,PPARγ,PIP1B,RBP-J,STAT3,STAT6,TIPE2 and TRAF6）.Immunofluorescence（IF）was used to detect C/EBPβexpression in AMs.4.Experiment methodsHE staining was performed for morphological analysis;Masson’s Trichrome staining and hydroxyproline（HYP）kit were used to determine fibrotic changes of lung tissue;Immunohistochemistry（IHC）and Western Blotting were used to detect EMT level in lung tissue and BEAS-2B cells and FMT level in lung tissue and fibroblasts;Flow cytometry was used to detect the proportions of F4/80⁺CD11c⁺and F4/80⁺CD206⁺AMs;IF was used to detect levels of F4/80,CD11c,CD206 and C/EBPβin AMs;RT-PCR was used to detect m RNA expressions of i NOS,Arg-1,TNF-α,IL-6,TGF-β,IL-10 and15 candidate genes-associated M2 macrophage polarization（AMPKα,BMP4,B7-H3,C/EBPβ,Fox O1,Herpub1,IRF4,KLF4,PPARγ,PIP1B,RBP-J,STAT3,STAT6,TIPE2and TRAF6）;ELISA was used to detect activities of i NOS and Arg-1 and contents of TNF-α,IL-6,TGF-β,IL-10 and IL-4 in BALF and supernatants of culture medium.Results1.TGF-βwas involved in CNTs promote AMs polarization toward M2subtype-mediated EMT in BEAS-2B cells and FMT in primary lung fibroblasts.1.1 Primary mouse AMs polarized toward M1 subtype after LPS+IFN-γstimulation（M1 polarization condition）and toward M2 subtype after IL-4 stimulation（M2polarization condition）.CD11c positive AMs,i NOS m RNA expression in AMs and i NOS activity in supernatant of culture medium were significantly increased under M1 condition.LPS+IFN-γup-regulated m RNA expressions and contents of TNF-αand IL-6.CD206 positive AMs,Arg-1 m RNA expression in AMs and Arg-1 activity in the supernatant of culture medium were significantly increased under M2 condition.IL-4 up-regulated m RNA expressions and contents of TGF-βand IL-10.1.2 CNTs exposure（M1 condition）for 6 h promoted M1 AMs polarization,while CNTs exposure（M2 condition）for 24 h promoted M2 AMs polarizationUnder M1 polarization condition,CNTs exposure for 6 h up-regulated F4/80⁺CD11c⁺AMs and m RNA expressions of i NOS,TNF-αand IL-6.After CNTs exposure for 6 h,activity of i NOS and contents of TNF-αand IL-6 in supernatant of culture medium were obviously increased.Under M2 polarization condition,CNTs exposure for 24 h up-regulated F4/80⁺CD206⁺AMs and m RNA expressions of Arg-1,TGF-βand IL-10.After CNTs exposure for 24 h,activity of Arg-1 and contents of TGF-βand IL-10 in the supernatant of culture medium were obviously increased.1.3 TGF-βwas involved in CNTs promote AMs polarization toward M2subtype-mediated EMT and FMT.CM experiments showed that CNTs-exposed AMs for 6 h did not change EMT and FMT.CNTs-exposed AMs for 24 h induced EMT in BEAS-2B cells and FMT in primary lung fibroblasts.Based on CM experiments in vitro,we found that TGF-βaggravated CNTs-caused EMT in BEAS-2B cells and FMT in primary lung fibroblasts.However,TGF-βinhibitor alleviated CNTs-caused EMT in BEAS-2B cells and FMT in primary lung fibroblasts.2.The role of AMs polarization in CNTs-induced pulmonary fibrosis2.1 CNTs induced lung inflammation at early stage and induced pulmonary fibrosis at late stage based on a mice modelCNTs exposure significantly up-regulated lung coefficient and HYP in lung tissues in a time-dependent manner.HE staining showed that CNTs exposure for 3 days induced obvious inflammatory cells infiltration,while CNTs exposure for 7 and 28 days induced thickening of alveolar wall.Masson’s Trichrome staining showed that CNTs exposure induced obvious collagen deposition of lung tissue in a time dependent manner.IHC and Western Blotting showed that CNTs exposure induced down-regulation of E-cad and up-regulation of Vimentin,α-SMA and CollagenⅠof lung tissue on days 7 and 28.2.2 CNTs exposure induced AMs polarization toward M1 subtype at early stage,while toward M2 subtype at late stageA significant increase in F4/80⁺CD11c⁺AMs and m RNA expressions of i NOS,TNF-αand IL-6 were observed on day 3 after CNTs exposure.Meanwhile,CNTs exposure up-regulated activity of i NOS and contents of TNF-αand IL-6 on day 3.A significant increase in F4/80⁺CD206⁺AMs and m RNA expressions of Arg-1,TGF-βand IL-10were observed on days 7 and 28 after CNTs exposure.CNTs exposure up-regulated activity of Arg-1 and contents of TGF-βand IL-10 on days 7 and 28.2.3 M2 AMs polarization was involved in SWCNT-induced pulmonary fibrosis IL-4 neutralization inhibited IL-4 release in BALF of mice.Furthermore,IL-4neutralization inhibited SWCNT-activated M2 AMs polarization and related cytokines（TGF-βand IL-10）secretion.In addition,IL-4 neutralization increased SWCNT-downregulated E-cad,as well as IL-4 neutralization reduced SWCNT-upregulated Vimentin,CollagenⅠandα-SMA of lung tissues.3.C/EBPβwas involved in SWCNT-promoted M2 AMs polarization3.1 SWCNT up-regulated C/EBPβexpression in AMs in vivoIn vivo experiments,SWCNT up-regulated C/EBPβ,KLF4,IRF4 and STAT6 at m RNA levels on day 28,thereinto,C/EBPβwas the most prominent genes.IF showed that SWCNT up-regulated C/EBPβat protein expression.Therefore,C/EBPβmaybe involved in SWCNT-induced M2 AMs polarization.3.2 Silencing C/EBPβreduced SWCNT-upregulated M2 AMs polarizationIn vitro experiments,under M2 polarization condition,SWCNT exposure for 24 h promoted C/EBPβprotein expression in AMs.After C/EBPβsi RNA treatment,SWCNT-upregulated F4/80⁺CD206⁺AMs and m RNA expressions of Arg-1,TGF-βand IL-10 were significantly decreased.Meanwhile,C/EBPβsi RNA reduced SWCNT-upregulated activity of Arg-1 and contents of TGF-βand IL-10 in supernatant of culture medium.Therefore,silencing C/EBPβreduced SWCNT upregulated M2 AMs polarization.3.3 C/EBPβsi RNA alleviated SWCNT-induced EMT and FMTCM experiments showed that E-cad expression was increased and Vimentin was decreased in BEAS-2B cells in SWCNT+C/EBPβsi RNA group compared to SWCNT group,indicating C/EBPβsi RNA alleviated SWCNT-induced EMT in bronchial epithelial cells.Furthermore,α-SMA and CollagenⅠprotein expressions were obviously decreased in primary lung fibroblasts in SWCNT+C/EBPβsi RNA group compared to SWCNT group,indicating C/EBPβsi RNA alleviated SWCNT-induced FMT in primary lung fibroblasts.ConslusionCNTs exposure promoted M1 AMs polarization along with lung inflammation at early stage,while promoted M2 AMs polarization along with pulmonary fibrosis at late stage.Thereinto,SWCNT showed the strongest capacity to induce M2 AMs polarization and pulmonary fibrosis among three types of CNTs.Additional experiments using IL-4neutralization showed that M2 AMs polarization was involved in SWCNT-induced pulmonary fibrosis.At last,we elucidated SWCNT promoted AMs polarization toward M2 phenotype-mediated pulmonary fibrosis via upregulating C/EBPβin vivo and vitro.