Reagents and antibodies
The rabbit anti-GBP1 polyclonal antibody (Catalog No. PA5-23509) was purchased from Life technologies. The following rabbit antibodies, including anti-iNOS (Catalog No. 13120), anti-TNF-a (Catalog No. 11948), anti-NF-κBp65 (Catalog No. 8242) anti-phospho-NF-κBp65 (Catalog No. 3033), anti-β-actin (Catalog No. 8457), anti-TLR4 (Catalog No. 14358), and the goat anti-rabbit secondary antibody (Catalog No. 7074) were purchased from Cell Signaling Technology (Boston, Mass, USA). APC-conjugated anti-mouse CD86 antibody was purchased from R&D Systems (Minneapolis, MN). Nuclear dye DAPI was purchased from Thermo Fisher Scientific (Waltham, MA).
Bacterial culture and infections
Virulent M. bovis Beijing strain was obtained from the China Institute of Veterinary Drug Control (CVCC, China). M. bovis was cultured in 7H9 Middlebrook media (Catalog No. BD271310, BD Biosciences) containing 10% OADC enhancement solution (Catalog No. BD 212352, BD Biosciences) and incubated continuously for a week at 37 °C to medium logarithmic period. BMDMs and RAW264.7 cells were infected with M. bovis (MOI 10) at 37°C with 5% CO2. Cells were washed three times with warm PBS to remove extracellular bacteria after 3 h.
Macrophage culture, induction, and RNA isolation
RAW264.7 cell line (Catalog No. BNCC354753) purchased from ATCC and cultured in Dulbecco's Modified Eagle Medium (Catalog No.10313021, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Catalog No.10099141C, Thermo Fisher Scientific). As described previously , BMDMs were isolated from C57BL/6 mice. BMDMs were cultured in Roswell Park Memorial Institutes 1640 Medium containing 20 ng/mL recombinant murine M-CSF (Catalog No. AF-315-02-100, Pepro Tech) and 10% FBS for 5-7 days to become adherent macrophages, and these cells were named M0 BMDMs. Subsequently, M0 BMDM was converted to M1 BMDM after induction by 100 ng/ml LPS and 50 ng/ml IFN-γ for 48 h. M0 BMDM was converted to M2 BMDM after induction by 10 ng/ml IL-4 and IL-13 for 48 h. Four sets of cell samples (M1 BMDMs, M2 BMDMs, M0 BMDMs, and M0 BMDMs infected with M. bovis for 6 h) for RNA sequencing, with three biological replicates per sample. RNeasy Mini kit (Catalog No.74104, Qiagen) was used to obtain total RNA with the manufacturer’s protocol. RNA samples were quantified and quality controlled with a Nanodrop Lite spectrophotometer (Thermo Fisher Scientific, USA). Finally, RNA samples with a spectral A260/A280 nm ratio between 1.8 and 2.0 and an A260/A230 nm ratio >1.5 were selected for analysis.
Analysis of differentially expressed mRNAs
As previously described [29, 30], the R package DESeq2 algorithm was used to identify differentially expressed genes and mRNAs for identifying transcripts from RNA sequence data. q-value < 0.05 and log2 FC > 1 indicate thresholds for differentially expressed genes.
As previously described [29, 30], pathway analysis was performed to identify significant pathways for differential genes based on KEGG. Fisher’s exact test and χ2 test were used to select significant pathways, and significance thresholds were determined by p-values.
GBP2b-mRNA co-expression correlation analysis
The differential mRNAs associated with GBP2b were screened by calculating the threshold of Pearson |R| > 0.95 between GBP2b and differential mRNAs, and then the pathway regulatory relationships involved in the differential mRNAs were used to establish the GBP2b-ComRNA-Pathway-Network, demonstrating the pathway mainly regulated by GBP2b.
Knockdown of GBP2b and TLR4 expression
Three siRNA sequences specifically targeting mouse GBP2b and mouse TLR4 were designed according to Gene Chem Co., Ltd. (http://www.genechem.com.cn), and siRNA sequences are shown in Table 1. Finally, the efficiency of GBP2b and TLR4 knockdown was examined using RT-PCR and Western blot 48 h after transfection, and the most efficient siGBP2b-3 and siTLR4 -3 were finally determined.
Overexpression of GBP2b
The full-length coding sequence of GBP2b (NM_010259.2, 1800 bp) was amplified by PCR from RAW264.7 cells cRNA, cloned into the pLVX-Puro vector, and then sequenced. The primers for GBP2b are as follows:
The PCR products were purified and cloned into the pLVX-Puro plasmid and then sequenced. The GBP2b overexpression plasmid (pLVX-GBP2b) and an empty vector (pLVX-Con) were transfected into RAW264.7 cells with Lipofectamine 3000 reagent (Catalog No. L3000001, Thermo Fisher Scientific). The protein expression of GBP2b was detected by Western blot to evaluate the overexpression efficiency of transfection.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using the RNeasy Mini kit (Catalog No.74104, Qiagen), and the concentration and integrity were detected by Nanodrop Lite spectrophotometer (Thermo Fisher Scientific, USA). Prime Script TM RT Master Mix (Catalog No. RR036B, Takara) was used for the reverse transcription of RNA to cDNA. RT-PCR analysis was performed using FastStart Universal SYBR Green (Catalog No. 4913850001, Takara) and Light cycler 480 RT-PCR instruments. The results were normalized to GAPDH, and quantification was performed on the 2-ΔΔCt of each sample/2-ΔΔCt of the Ctrl method. The primers are listed in Table 2.
Animal and M. bovis infection
C57BL/6 mice (male, aged 6–8 weeks) were purchased from Yangzhou University (Yangzhou, China) and raised in a level III biosafety facility (Nanjing Institute for Food and Drug Control, Nanjing). The mice were divided into two groups of 6 mice each. In one group, 200 CFU of M. bovis per mouse were given intranasally. The other group was inoculated with the same dose of sterile PBS. After 6 weeks, the spleens and lungs of both groups of mice were collected to detect GBP2b protein and mRNA levels.
Colony-forming unit (CFU)
RAW264.7 cells were transfected with GBP2b silencing vectors (siGBP2b), GBP2b overexpression vectors (pLVX-GBP2b), and the respective negative control vectors (siCon and pLVX-Con) and then infected with M. bovis (MOI 10) at different time points. RAW264.7 cells were subsequently washed three times with warm PBS and lysed with sterile 0.2% Triton X100/PBS for 5 min. After serial dilutions of cell lysates, 100 µL of homogenate was evenly spread in a 7H11 solid medium (Catalog No. BD 283810, BD Biosciences) and left at 37 °C for 3-4 weeks. Bacterial CFUs were counted to assess the bactericidal capacity of the cells in the different treatment groups.
The culture supernatant under each culture condition was collected, and the concentration of nitrite in the supernatant was determined using Griess reagent (Catalog No. 13547, Cell Signaling Technology) with the manufacturer’s instructions. Finally, the absorbance of the samples was obtained at 540 nm on a microplate reading instrument.
Immunofluorescence analysis was performed to identify the localization of NF-κBp65 in RAW264.7 cells transfected with the GBP2b silencing vector (siGBP2b), the GBP2b overexpression vector (pLVX-GBP2b), and the corresponding negative control vectors (siCon and pLVX-Con) and then infected with M. bovis for 24 h. In brief, cells were fixed with 4% paraformaldehyde for 25 min, washed three times with PBS, and then permeabilized for 10 min after containing 0.2% Triton X-100/PBS. Cells were subsequently blocked with 3% bovine serum albumin for 1 h at room temperature, and then anti-NF-κBp65 was incubated overnight at 4 °C. Finally, cells were incubated with Alexa Fluor 647-conjugated anti-rabbit antibody for 1 h, and nuclear staining was performed using DAPI. The cells were visualized immediately with an OLYMPUS microscope (Suzhou Jing Kai Instrument and Equipment Co., Ltd., Suzhou, China). Approximately 100 cells are used to calculate the fluorescence intensity. The Image J software was used for the analysis.
BMDMs and RAW264.7 cells from different treatment groups were collected and incubated for 30 min with an APC-conjugated anti-mouse CD86 antibody (1:200) or CellROX reagent (a highly ROS-specific probe, Catalog No. C10488, Thermo Fisher Scientific), respectively. CD86 is used as a marker for M1 macrophages. CellROX is used to detect intracellular ROS. Finally, the cells were resuspended at 500 μL in PBS and FACS caliber flow cytometry (BD, Franklin Lakes, USA). Data were analyzed by Flow Jo software.
After different treatments, BMDMs, RAW264.7 cells, mouse lung, and spleen tissues were washed three times with cold PBS and lysed with RIPA buffer (Catalog No. P0013C, Beyotime) containing protease inhibitors. Protein concentration was quantified by a BCA protein analysis kit (Catalog No. P0012S, Beyotime), and protein was transferred to polyvinylidene fluoride membranes (Catalog No.88518, Thermo Fisher Scientific,) following separation on SDS-PAGE gels. The membranes were incubated with primary antibodies overnight at 4 °C and then washed three times with TBST buffer. Subsequently, they were exposed to peroxidase-coupled secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized with an enhanced chemiluminescence solution. Image J software is used to analyze protein expression and β-Actin acts as load control.
The gene expression was calculated by RSEM (v1.2.12) https://github.com/ deweylab/RSEM). The heatmap was drawn by heatmap (v1.0.8) (https://cran.r-project.org/web/packages/pheatmap /index.html) according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2 (v1.4.5) (http://www.bioconductor.org/ packages/release/bioc/html/DESeq2.html) with a Q value ≤ 0.05. KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression genes was performed by Pyper (https://en.wikipedia.org/wiki/ Hypergeometric_distribution) based on the Hypergeometric test. GraphPad Prism 8.0.1 was applied to analyze all the experimental data. Data are presented as the means ± standard error or mean ± standard deviation. P< 0.05 was considered significant. All assays were performed on at least two or three separate occasions in triplicate each time.