The mouse anti-Map-LC3 antibody (sc-376404), the rabbit polyclonal anti-mouse AIM2 antibody (sc-137967) and the goat polyclonal antibody TMEM173 (M-12) (sc-241049) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-mouse β-actin (AP0060) was obtained from Bioworld Technology (Nanjing, Jiangsu, China). The rabbit Anti-NAK/TBK-1 antibody (ab40676) and rabbit anti-NAK/TBK-1 (phosphor S172) antibody (ab109272) were obtained from Abcam (Cambridge, UK). The rabbit TMEM173 polyclonal antibody (19851-1-AP) and rabbit IRF3 polyclonal antibody (11312-1-AP) are from Protein Tech (Wuhan, Hubei, China). The phosphor-IRF3 (Ser396) (4D4G) and the Acetylated-Lysine Antibody (9441) were from Cell Signaling Technology (Boston, Mass, USA). The goat anti-rabbit secondary antibody, rabbit anti-mouse secondary antibody and donkey anti-goat secondary antibody were obtained from Santa Cruz Biotechnology, Beijing ZSGB Biotechnology and Beijing Cowin Biotechnology (Beijing, China), respectively. The BX-795 (S1274) was from Selleckchem. The Fast Protein Precipitation and Concentration Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Reagents and apparatus used in immunoblotting assays were obtained from Bio-Rad (Hercules, CA, USA). The mouse interferon-β ELISA kit (CSB-E04945m) was from Cusabio (Wuhan, Hubei, China).

Mouse macrophage cell lines J774A.1 were obtained from Cell Culture Center, Xiehe Medical University (Beijing China), and were cultured as described before (Liu et al., 2016). Mouse bone marrow derived macrophages (BMDMs) were isolated from femurs of 6–8 week old female C57BL/6 mice as described previously (Liu et al., 2016), and cultured in circular cell culture dish (Corning, New York, USA) for 7 days in RPMI1640 (Hyclone, Logan, UT, USA) supplemented with 10 ng/ml M-CSF (Pepro Tech), 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 ug/ml streptomycin and 100 U/ml penicillin (Gibco).

Virulent M. bovis Beijing strain was obtained from China Institute of Veterinary Drug Control (CVCC, China). Bacteria were cultured from frozen stocks in 7H9 Middlebrook media (BD Biosciences) containing albumin- dextrose-catalase (ADC) enrichment solution and 0.05% Tween-80 (Difco) and grown to mid-logarithmic phase for 1 week at 37°C. J774A.1 and BMDMs were infected with M. bovis (MOI = 10:1) at 37°C with 5% CO₂. Cells were washed three times with warm PBS to remove extracellular bacteria after 2 h. All experiments were performed in triplicate on three different occasions.

Mouse IFI204-targeting siRNA oligonucleotide was obtained from Qiagen (Valencia, CA, USA). For siRNA transfection, cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well, and then transfected with siRNA oligonucleotides (50 nM) using 3 μL HiperFect Transfection Reagent (Qiagen, Valencia, CA, USA). The decrease in IFI204 expression was analyzed by qRT-PCR and western blot analysis.

The reagents for immunohistochemical (IHC) staining (TAHC03D) were obtained from BIOTnA (Sanming, Fujian, China), and the lung tissues from C57BL/6J mouse infected with M. bovis (CFU = 10⁴) for 4 weeks were stained as per manufacturers' instructions.

NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833) was obtained from Thermo Scientific, the protein from nucleas and cytoplasm was extracted as per manufacturers' instructions.

Total RNA was extracted using SV Total RNA Isolation System (Promega, Madison, WI, USA), and the concentration and integrity were detected by NanoDrop 2000 machine (Thermo SCIENTIFIC, Waltham, MA, USA). Reverse transcription of 100 ng RNA was performed by the Revert Aid first-strand cDNA synthesis Kit (Fermentas, Glen Burnie, MD, USA) according to the manufacturer's instructions, and the integrity and concentration of cDNA were detected by NanoDrop 2000 machine to confirm same amount of cDNA used for different samples. Quantitative PCR was performed using the DNA Engine Opticon TM2 fluorescence detection system (MJ Research Inc., Waltham, MA, USA) and SYBR Green Master Mix (Bio-Rad). The special gene primers pairs which cited from the publications (Yang et al., 2013) are shown in Table 1. Quantitative PCR data were analyzed using the comparative CT method (2^−ΔΔCT) according to the application guidelines of fluorescence quantitative PCR from BIO-RAD. All samples were analyzed in triplicate.

Total cell proteins were extracted using a protein extract kit (Fast Protein Precipitation and Concentration Kit, Wuhan Boster Biotech, Wuhan, China), and then were mixed with 5xSDS sample buffer. The mixtures were separated by different SDS-PAGE (8–15%) according to the molecular weight and analyzed by immunoblot as described previously (Liu et al., 2016). The image J software was used to analyze the intensity.

ELISA for IFN-β was performed with the macrophage cell culture supernatants by using mouse interferon-β ELISA kit as per manufacturers' instructions.

Ten microliter of a log-phase M. bovis culture were transferred into a 15-mL conical tube. Mycobacterial cultures were centrifuged at 3,000 rpm for 5 min, the supernatant fraction was carefully removed and the pellet washed twice with 10 mL of 1x PBS. This was centrifuged at 3,000 rpm for 5 min and the supernatant fraction removed and discarded. The pellet was resuspended in 1 mL of 1x PBS and the cell suspension transferred into a 1.5-mL microcentrifuge tube. 10 μl of Alexa 488 caboxylic acid succinimidyl ester stock solution was added to the tube to make a final concentration of 10 mg/ml. The tube was wrapped with foil and incubated at 37°C for 45–60 min on a shaker. Mycobacteria were pelleted by centrifugation at 10,000 rpm for 3 min at room temperature (RT). The supernatant was removed and washed twice with 1 mL of 1XPBS. The mycobacterial pellet was resuspended in 6 mL of complete DMEM. All reagents used for fixing, washing and blocking the cells were purchased from Beyotime (Beyotime Institute of Biotechnology, China). Cells were grown on poly-lysine-coated coverslips. After treatment, cells were fixed in an Immunol-staining fix solution (Beyotime) for 10 min at RT and washed three times for 5 min with PBS. Then they were blocked for 1 h at RT. Mouse anti-LC3B (1:200), rabbit anti-p-TBK-1 (1:200), rabbit anti-NAK (1:200), rabbit anti-IRF3 (1:200), rabbit anti-IFI204 (1:100) and goat anti-TMEM173 (1:50) were added, respectively, and incubated at 4°C overnight and washed three times with PBS for 5 min. Goat anti- rabbit IgG Alexa Fluor 594 (1:200), FITC-conjugated antibody (1:200), and donkey anti-goat IgG Alexa Flour 647 (1:200) were added and incubated at 37°C for 1 h and washed three times with PBS for 5 min. DAPI (1:10) was added and incubated at RT for 5 min and washed three times with PBS for 5 min. The macrophages were finally mounted with glycerin buffer and examined immediately with an OLYMPUS microscope.

Cells were infected with M. bovis and washed twice with cold 1XPBS. Ice-cold RIPA or NP40 buffer with protease inhibitors was added and incubated at 4°C for 30 min on a rocker, to lyse the cells. Lysates were by micro-centrifuging at 13,200 rpm for 20 min in 4°C. The supernatant fraction was immediately transferred to a new centrifuge tube and the pellet was discarded. Cell lysate was incubated with 30 μl protein A or G agarose/sepharose beads (50%) at 4°C for 30 min on a rocker. Protein A or G beads were removed by centrifugation at 13,200 rpm at 4°C for 1 min and supernatant was transferred to a fresh centrifuge tube. A 30 μl aliquot od 50% protein A or G agarose/sepharose slurry was added with a specific antibody. After an overnight incubation at 4°C on a rocker the beads were washed 3–4 times for 10 min each with RIPA or NP40 buffer. Agarose/sepharose beads were resuspended in 30 μl 1X sample buffer and mixed gently. The agarose/sepharose beads were then boiled for 5min to dissociate the immune-complexes from the beads and spun down and the supernatants were used for SDS-PAGE and Western blot analysis.

J774A.1 and BMDMs cells were plated in 12-well plates (5 × 10⁵ cells/well). Cells were infected with 5 × 10⁶ mycobacteria per cells for 3 h at 37°C, washed three times with warm PBS. The CFUs were determined by plating 100 μL of serial dilutions onto plates containing Middlebrook 7H11 agar, supplemented with ADC enrichment solution and 0.05% Tween-80 (Difco).

All assays were performed on two or three separate occasions in triplicate each time. Results are expressed as means ± S.D. All data comparisons were performed using one-way ANOVA followed by post-hoc Tukey's test or Student's t-test. SPSS software (Chicago, IL, USA) was used, and P < 0.05 was considered significant.

All protocols and procedures were performed according to the Chinese Regulations of Laboratory Animals—The Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of People's Republic of China) and Laboratory Animal Requirements of Environment and Housing Facilities (GB 14925–2010, National Laboratory Animal Standardization Technical Committee). The license number associated with their research protocol was 20110611–01 and the animal study proposal was approved by The Laboratory Animal Ethical Committee of China Agricultural University.

Previous studies have demonstrated that type-I interferon could be induced by mycobacterium infection (Pandey et al., 2009; Manzanillo et al., 2012). STING-dependent IRF3 phosphorylation mediated by the cytosolic DNA sensor plays an important role in this process. Subsequently, the phosphorylated IRF3 (p-IRF3) translocates into the nucleus leading to IFN-β release (Dey et al., 2015). To examine whether murine macrophages respond similarly to M. bovis, J774A.1 cells and BMDMs were infected with a virulent strain of M. bovis isolated from cattle at a multiplicity of infection (MOI) of 10 and evaluated in a time course experiment (0, 4, and 24 h). Production of IFN-β was measured by ELISA (Figures 1A,B). We observed a time-dependent induction of IFN-β release. Western blot (WB) for total protein and phosphorylated counterparts were performed to investigate IRF3 responses during M. bovis infection. As expected, the IRF3 protein was phosphorylated during M. bovis infection (Figure 1C). Immunofluorescence (IF) microscopy showed that IRF3 translocated from cytoplasm to the nucleus at 24 h post-infection in J774A.1 cells (Figure 1D). Furthermore, the relative concentrations of IRF3 clearly increased in the nucleus and reduced in the cytoplasm of infected J774A.1 cells at 4 and 24 h-post-infection (Figure 1E). These results confirmed that IFN-β expression is induced in murine macrophages exposed to a virulent strain of M. bovis, and the release of IFN-β was dependent on IRF3 activation and its nuclear translocation.

To test whether IFI204 expression changes during M. bovis infection, we measured IFI204 mRNA level using q-PCR, at different infection time points. We observed that the mRNA expression of IFI204 was upregulated in both J774A.1 (Figure 2A) and BMDMs (Figure 2B). IFI204 protein showed a sustained increase in both cell types and peaked at 24 h post-infection (Figures 2C,D). The protein production of IFI204 showed a behind its mRNA expression may attribute to the asynchronous expression of the gene and protein. The results also showed the gene expression of IFI204 decreased after 48 h in J774A.1 but at 24 and 48 h in BMDM cells. One reason for this difference may be related to more rapid proliferation by J774A.1 cells. Another factor is M-CSF within the myeloid cell culture medium affects the IFI204 expression (Dauffy et al., 2006). IFI204 expression in the lung lesions of mice infected with M. bovis showed significantly higher IFI204 expression in the sections with granulomas (Figure 2E). We then demonstrated that IFI204 was present in both the cytoplasm and the nucleus and the concentrations of IFI204 were higher in the nucleus (Figure 2F). Furthermore, we showed that the relative intensity of IFI204 was enhanced in both cytoplasm and nucleus at 24-h post-infection. Taken together, IFI204 is induced by M. bovis infection in murine macrophages. It was also expressed in the lungs of M. bovis infected mice.

To examine the role of IFI204 in IFN-β release in response to M. bovis infection, we applied IFI204 specific small interfering RNA (siRNA) to knock down the expression of IFI204 in the BMDM infection model. The results showed that IFI204 expression decreased by 55 percent post-blocking (Figure 3A). IFN-β expression measured by qPCR and ELISA showed that both mRNA (Figure 3B) and protein (Figure 3C) expression decreased in IFI204 knock-down groups. Gene expression of TNF-α and IL-10 did not show any significant changes but an increase in IL-1β levels were observed (Figure 3D). We have recently shown that AIM2 plays a critical role in IL-1β release and it inhibits the STING-dependent pathway during M. bovis infection in macrophages (Liu et al., 2016). We hypothesized that IFI204 conjugated competitively with M. bovis to inhibit the AIM2-dependent IL-1β production. To test this hypothesis, we examined the co-localization of M. bovis and AIM2 after IFI204 knocked down. M. bovis-AIM2 co-localization increased moderately (Figure 3E). Taken together, these results suggest that IFI204 plays an important role in IFN-β release during M. bovis infection in murine macrophage model.

As shown in Figure 2F, IFI204 expression increased in the cytoplasm and the nucleus during M. bovis infection. Furthermore, the IFI204 protein levels in the respective cellular compartments were confirmed by WB (Figure 4A). Results show that the expression in both cellular sub-compartments increased in a time dependent manner during macrophage infection. The translocation of IFI16 from cytoplasm to nucleus is regulated by the acetylation of nuclear IFI16 during viral infections (Ansari et al., 2015). We found that IFI204 was acetylated in macrophages during M. bovis infection and the acetylated protein was distributed in cytoplasm but not in the nucleus (Figure 4B). We next demonstrated that cytosolic IFI204 co-localized with M. bovis (Figure 4C) in 42% infected macrophages. STING is the adapter protein for IFI16 which is known to be required for TBK1-IRF3-dependent IFN-β responses to viruses and DNA (Unterholzner et al., 2010). To identify if STING molecule was recruited by Ifi204 during M. bovis infection, we used IF to observe the co-localization between them at 24h post-infection in BMDMs (Figure 4D). Co-immunoprecipitation (co-IP) experiments showed that STING was present in IFI204 immunoprecipitates, suggesting existence of a IFI204-STING conjugate molecule in J774A.1 cells (Figure 4E, left column). No interaction was observed between ASC and IFI204 in the infected cells (Figure 4E, right column). This result further confirmed our hypothesis that IFI204 conjugates competitively with M. bovis to inhibit the AIM2-dependent IL-1β production. To examine the influence of IFI204 on downstream signaling pathway, we performed siRNA to down-regulate IFI204 expression and then tested the phosphorylation of TBK1 and IRF3 by WB in J774A.1-M. bovis infection model. We observed a decrease in STING-dependent signaling molecules (Figure 4F). Taken together, we conclude that IFI204 first undergoes acetylation and then translocates from nucleus into cytoplasm to recruit STING for activation of TBK1-dependent IRF3 nuclear translocation and IFN-β release.

TBK1 has been shown to play an important role in autophagy activation during cytosolic bacterial infection (Pilli et al., 2012). The dsDNA sensor cGAS has been shown to be associated with induction of autophagy and co-localization between LC3 and Mtb in macrophages (Watson et al., 2015). To test the role of IFI204 in autophagy, we examined the LC3 expression in BMDMs transfected with IFI204 targeting siRNA and then infected with M. bovis. We observed that LC3-IIexpression decreased when IFI204 was knocked down (Figure 5A). Similar results were observed when LC3-puncta were counted (Figure 5B). Next, we tested whether IFI204 also had an effect on the co-localization between M. bovis and LC3. Absence of IFI204 significantly reduced the association of LC3 with M. bovis (Figure 5C).

Type I interferons (IFN) are pleiotropic proteins with anti-proliferative, antiviral, and immunomodulatory activities. It is widely believed that IFN-β is an adverse factor for host survival, but advantageous to mycobacterial survival. Multiple investigations have demonstrated that induction of autophagy inhibits mycobacterium replication in macrophages. Furthermore, cGAS, which has been shown to induce type-I interferon and activate autophagy during Mtb infection, is necessary for mycobacterium control. To examine the role of IFI204 in M. bovis survival, we infected BMDMs and J774A.1 deficient in IFI204 and quantified intracellular mycobacterium replication (Figure 6). No significant differences were observed in M. bovis replication between knockdown and untreated cells. These results suggest an important role of IFI204 in IFN-β release and autophagy activation in murine macrophages during M. bovis infection.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.