Cell culture and measurement of ROS production
THP-1 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA). The cells were resuspended in fresh media at a concentration of 1 × 105/mL and pretreated with or without TSLP (PEPROTECH, New Jersey, USA) for 2 h. ROS production was measured with a dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma–Aldrich, MO, USA) assay by flow cytometry according to the methods described in previous studies [39, 40]. Following treatment with TSLP, THP-1 cells were probed with 1 mM DCFH-DA and then washed with PBS once for immediate detection of dichlorofluorescein (DCF).
Mitochondrial complex activity
The mitochondrial fraction was isolated from THP-1 cells using a mitochondrial isolation kit, and the activities of complex I and complex II/III in whole cell lysates or isolated mitochondria were measured using a microplate assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.
Measurement of ΔψM and transmission electron microscopy
THP-1 cells were treated with TSLP for 8 h, and JC-1 dye (Invitrogen, Waltham, USA) was used to measure the mitochondrial membrane potential by LSR II flow cytometry (Becton Dickinson, San Jose, CA). JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (525 nm) to red (590 nm). Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. After TSLP treatment, the cells were prepared for JEM-2000EXII transmission electron microscopy (JEOL Ltd. Tokyo, Japan) to observe the morphology of mitochondria and mitophagy.
Preparation of mitochondrial DNA (mtDNA) and relative quantification of mtDNA fragments
The total mtDNA of THP-1 cells were isolated after 8-h TSLP stimulation by a mitochondrial DNA isolation kit (BioVision, Milpitas, USA) following the manufacturer’s instructions. Two primer sets specific for the mitochondrial ribosomal 16SRNA, mtDNA-79 and mtDNA-230, were designed as described in a previous study [41, 42]. The 79-bp fragment (mtDNA-79) represents mitophagy-mediated mtDNA generated by enzymatic cleavage in apoptotic cells. In contrast, the 230-bp fragment (mtDNA-230) corresponds to mtDNA released by non-apoptotic types of cell death (i.e., necrosis) or active secretion [41,42,43]. Quantitative analysis of mtDNA fragments was performed by quantitative real-time PCR, and the data were evaluated for mtDNA fragments relative to housekeeping gene (ACTB) expression.
To detect mitophagy, we used the Mitophagy Detection Kit (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) according to the manufacturer’s instructions. Briefly, THP-1 cells were incubated with serum-free RPMI containing 100 nM mitophagy dye at 37 °C for 30 min. After washing and discarding the supernatant, the cells were stimulated with 40 ng/mL TSLP in complete medium for 12 h. Mitophagy dye fluorescence was detected by an LSR II flow cytometer (Becton Dickinson, San Jose, CA) with a PerCP Cy5.5 channel. The data were analyzed using FCS Express 4 Flow Research (De Novo Software, California, USA).
Quantitative real-time reverse transcription polymerase chain reaction (qRT–PCR) analysis of respiratory chain and mitochondrial biogenesis factors
The gene expression of mitochondrial copy numbers, respiratory chain complexes I–V, including NADH dehydrogenase, subunit 1 (MT-ND1, MT complex I), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA, MT complex II), mitochondrial cytochrome b (MT-CYTB, MT complex III), cytochrome c oxidase I (COXI, MT complex IV), ATP synthase, H + transporting, mitochondrial Fo complex, subunit F6 (MT-ATP6, MT complex V), and mitochondrial proton carrier: uncoupling protein 2 (UCP2) were detected to investigate mitochondrial involvement. After treatment with TSLP for different durations, total RNA (1 μg) was reverse transcribed by SuperScript II using an anchored oligo-dT primer as instructed by the manufacturer (Invitrogen, Carlsbad, CA, USA), and the resulting DNA was used as template for qRT–PCR. The primer sequences and qRT–PCR were performed as described in our previous work [44, 45].
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed as described previously with minor modifications . Primers and probes were designed to analyze the proximal promoter regions relative to the transcription start site of the SDHA gene (− 2975/− 2577, including the AP-1 binding site) according to MatInspector (Genomatix Software, München, Germany). The primer sequences were as follows: forward: 5′-CTGACCACACTACCTCAGCA and reverse: 5′-CAGTGCTTGCTTCTTGGTGA. All TaqMan reagents were purchased from Applied Biosystems. The relative intensities of the amplified products were normalized to the input DNA levels.
THP-1 cells were treated with TSLP for 2, 4, 6 or 8 h and analyzed by western blotting with anti-MTCO2 antibody (Abcam), anti-5′-AMP-activated protein kinase (anti-AMPK, Cell Signaling, Massachusetts, USA), anti-phospho-AMPK (Cell Signaling Technology, Danvers, MA, USA), anti-PINK1 (Cell Signaling Technology, Danvers, MA, USA), anti-LC3 (MBL, Nagoya, Japan), anti- phospho-Parkin (Ser65) Antibody #36,866 (Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western blotting analysis was performed as described in our previous work .
Cultured THP-1 cells were treated with or without TSLP and fixed on glass slides. The cell slides were washed twice with PBS and permeabilized with 0.5% Triton for 5 min. These slides were then incubated in blocking solution (5% normal goat serum and 1% bovine serum albumin in PBS) for 30 min. After being blocked, the slides were incubated with PINK1, LC3, phospho-Parkin (Ser65) Antibody #36,866 (Cell Signaling Technology, Danvers, MA, USA) or phospho-Ubiquitin (Ser65), Cat. No. ABS1513-I (Merck Millipore, Darmstadt, Germany) antibodies, followed by Alexa Fluor 488-labeled and Alexa Fluor 568-labeled MitoTracker mitochondrial tracking dye for 1 h. After washing, 4′,6′-diamidino-2-phenylindole (DAPI) (300 nM; Invitrogen) dissolved in PBS was used as a nuclear staining dye, and the slides were mounted on Vectashield (Vector Laboratories Inc.), covered with glass coverslips, and imaged using an LSM 700 microscope (Carl Zeiss Microscopy, Gottingen, Germany).
Enzyme-linked immunosorbent assay (ELISA)
After 2 h of TSLP pretreatment, THP-1 cells were exposed to phorbol 12-myristate 13-acetate (PMA, 10 ng/mL, Sigma–Aldrich), LPS (10 ng/mL, Sigma–Aldrich) and IFN-γ (10 ng/mL, PEPROTECH) to induce M1 polarization or exposed to PMA and IL-4 (10 ng/mL, PEPROTECH) to induce M2 polarization. After being treated with or without PMA/LPS/IFN-γ or PMA/IL-4, the cell-free media were collected at 48 h, and CXCL10, TNF-α, IL-6, IL-1β, CCL-1, and CCL-22 were measured by ELISA (R & D Systems, Minneapolis, MN). CCL-22 concentrations in THP-1 cells and PMA/IL-4-primed THP-1 cells with or without the mitophagy inhibitor Mdivi-1 were also analyzed by ELISA.
THP-1 cells were transduced using shRNA lentiviral particles at a multiplicity of infection (MOI) of 1. A MOI is the number of transducing lentiviral particles per cell. All shRNA lentiviral particles were purchased from the RNA technology platform and gene manipulation core of Academia Sinica. The lentivirus encoded either a nontargeted (nt) shRNA (TRCN0000208001) or a shRNA directed against PINK1 (#TRCN0000007101, #TRCN0000007097, and #TRCN0000199446). A nontargeting (nt) shRNA (#TRCN0000208001) or shRNA targeting SHDA (#TRCN0000028085, #TRCN0000028093, and #TRCN000028118) was used to investigate whether SHDA was involved in TSLP-induced mitophagy. PINK1-knockdown cells were pretreated with TSLP for 2 h and then polarized to form M2 macrophages with PMA (20 ng/mL) and IL-4 (20 ng/mL). The supernatants were collected, and the CCL-22 concentration was measured after 48 h by ELISA.
GraphPad Prism (Version 5, GraphPad Prism Software, Los Angeles, CA, USA) and IBM SPSS statistical software (Version 19, IBM Company, Armonk, NY, USA) were used for statistical analysis. The Mann–Whitney U test was used for pairwise comparisons between samples with and without TSLP treatment. The immunoreactivities of mitophagy-related proteins in western blot analysis were normalized to those of the total proteins. The western blot results were analyzed by the Wilcoxon signed-rank test. Multiple nonparametric comparisons to assess the effects of TSLP on chemokines were analyzed by the Kruskal–Wallis test with post hoc analysis. Statistically significant differences were considered if a p value < 0.05.