Bone marrow-derived macrophages (BMDMs) were derived from murine femur–tibia bone marrow and prepared as previously described (27). In summary, isolated BMDMs were incubated at 5% CO₂, 37°C in complete Iscove’s modified Dulbecco’s medium (IMDM; Gibco, Grand Island, NY, USA) supplemented with 12.5 ng/ml of recombinant mouse M-CSF Protein (R&D Systems, Minneapolis, MN, USA), 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% MEM Non-Essential Amino Acids (MEM NEAA), and 1% penicillin/streptomycin (P/S). After 3 days, 5 ml of fresh same medium was added, and the cells were cultured under the same conditions for 3 days and then used in the experiment. For the treatment of bacteria and reagents, medium (hereafter referred to as treat medium) was supplied to IMDM with 2% FBS, 1% MEM NEAA, and 1% sodium pyruvate without antibiotics.

MH-S mouse alveolar macrophage (AM) cells purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 (WELGENE, Gyeongsan, Republic of Korea) supplemented with 10% FBS, 50 μM of 2-mercaptoethanol, and 1% P/S at 37°C in 5% CO₂ conditions. Treat medium was supplied to RPMI 1640 with 2% FBS and 50 μM of 2-mercaptoethanol without antibiotics.

Murine primary AMs were cultured as previously described (28). Briefly, 8- to 15-week-old male mice with C57BL/7 background were anesthetized, and bronchoalveolar lavage fluid (BALF) was obtained by flushing 1 ml of cold Dulbecco’s phosphate-buffered saline (D-PBS, WELGENE, Gyeongsan, Korea) containing 1 mM of EDTA. Collected BALFs from each mouse were pooled and centrifuged with 4°C and 250 relative centrifugal force (RCF). After the supernatant was discarded, pellets were dissolved with red blood cell (RBC) lysis buffer and incubated at room temperature for 10 min. After being washed twice with D-PBS, AMs were dissolved with DMEM high-glucose medium containing 10% FBS, 1% P/S, 1% HEPES, and 1% sodium pyruvate. AMs were seeded on a 48-well plate, 200 μl per well, with a concentration of 5 × 10⁵ cell/ml. After 12 h, the time to stabilize cells after attaching to plate, intracellular bacterial growth assay was performed with the same method of BMDMs and MH-S cells. Treat medium was supplied to DMEM high-glucose medium with 2% FBS, 1% HEPES, and 1% sodium pyruvate without antibiotics.

The following reagents were used. Mouse recombinant IFN-β (PBL Biomedical Laboratories, Piscataway, NJ, USA; Cat# 12401-1). Ultrapure lipopolysaccharide from E. coli O111:B4 (LPS; InvivoGen, San Diego, CA; Cat# tlrl-eblps), N ^G-Nitro-l-arginine methyl ester (l-NAME; Sigma Aldrich, St. Louis, MO, USA, Cat# 51298-62-5), and BX795 (InvivoGen, San Diego, CA, USA, Cat# tlrl-bx7).

WT, Ifnar ^−/−, Inos ^−/−, and Nod2 ^−/− mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Female mice aged 8–10 weeks were used for animal experiments. Animal experiments were performed under protocols approved by the Institutional Animal Care and the Use Committee of Chonnam National University (Approval No. CNU IACUC-YB-2017-56 and 2019-31).

Bacterial culture of the isogenic rough variant of M. abscessus ATCC19977T (Manassas, VA, USA) were prepared as previously described (29). Briefly, the bacterium was cultivated in 7H9 broth supplemented with 0.5% glycerol, 10% oleic acid, dextrose, albumin, and catalase (OADC; BD Biosciences, San Jose, CA, USA). For seed culture, 1 ml of frozen bacterial stock (3 × 10⁸ CFU) was added in 10 ml of culture medium, and the cells were allowed to grow for 7 days at 37°C with shaking at 130 rpm. For main culture, the entire cells from seed culture were added to 200 ml of culture medium and incubated for three more days. The cells were centrifuged at 4,500 RCF and washed three times with D-PBS. For single-cell suspension, the cells were dissolved in D-PBS and sonicated for 1 s at 40 kHz, 100 W. The cells aggregated by sonication were removed by passing through 40-μm mesh. This process was repeated three times. The seed lots dissolved in D-PBS (contained 40% glycerol) were kept at −80°C until use. In all in vitro experiments, the number of macrophages to the number of bacteria was set as 1:25 multiplicity of infection (MOI). This infection dose was determined in our previous study (16). Except the assessment of TBK1 phosphorylation (Western blotting), extracellular MAB was washed out after 1 h post infection, and cells were incubated with gentamicin (Sigma Aldrich, St. Louis, MO, USA) containing fresh medium for indicated times in all experiments

BMDMs and MH-S cells were seeded on a 48-well plate, 200 μl per well, with a concentration of 1 × 10⁶ cell/ml. After 12 h, the time to stabilize cells after attaching to plate, MAB diluted in treat medium was infected for 200 μl per well with MOI 25. After 1 h, which is a sufficient time for phagocytosis, cell culture supernatant was washed out by D-PBS and replaced with fresh treated medium containing 100 μg/ml of gentamicin. Cell culture supernatant was washed out by D-PBS on indicated time, and attached cells were lysed with 100 μl of 1% Triton X-100. The bacterial colony-forming unit (CFU) of cell lysate was determined by the spread plate technique, as follows. The cell lysate or lung lysate (in vivo) samples containing bacteria were diluted with D-PBS as proper detectable levels. Fifty microliters of diluted samples was uniformly spread through glass beads on the Middlebrook 7H10 agar (Difco, Detroit, MI, USA) plate containing ampicillin, and plates were incubated for 96 h at 37°C. The number of colony was counted and converted to log value. The CFU value of the tissue sample was normalized to tissue weight (g).

BMDMs and MH-S cells were seeded on a 6-well plate, 2 ml per well, with a concentration of 1 × 10⁶ cell/ml. After 12 h, the time to stabilize cells after attaching to plate, infection and reagent treatment were performed with each indicated conditions. RNA was extracted using 1 ml of easy-BLUETM Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). cDNA was synthesized from 1 μg of RNA using ReverTra Ace^® qPCR RT Master Mix (TOYOBO Biotechnology, Osaka, Japan). Real-time qPCR samples were prepared using the SYBR Green PCR Kit (Qiagen GmbH, Hilden, Germany). The primer sequences were as follows: iNOS; sense, 5′-GCATTGGAAGTGAAGCGTTTC-3′ and antisense, 5′-GGCAGCCTGTGAGACCTTTG-3′. IFN-β; sense 5′-ATGAACTCCACCAGCAGACAG-3′, and antisense, 5′-ACCACCATCCAGGCGTAGC-3′. CRAMP; sense 5′-AAGGAACAGGGGGTGGTG-3′ and antisense, 5′-CCGGGAAATTTTCTTGAACC-3′. NOX2; sense 5′-ACTCCTTGGAGCACTGG and antisense 5′-GTTCCTGTCCAGTTGTCTTC-3′. β-Actin; sense 5′-AGGCCCAGAGCAAGAGAG-3′ and antisense, 5′-TCAACATGATCTGGGTCAT-3′. PCR was conducted by a Rotor-Gene Q real-time PCR system (Qiagen) using a two-step protocol of 40 cycles of 95°C for 10 s followed by 60°C for 45 s. Normalized gene expression levels were indicated as the ratio between the mean value for the target gene and that for the β-actin.

BMDMs were seeded on a 12-well plate, 1 ml per well, with a concentration of 1 × 10⁶ cell/ml. After 12 h, the time to stabilize cells after attaching to plate, infection and reagent treatment were performed with each indicated conditions. Cells were lysed using lysis buffer containing Nonidet P‐40, complete protease inhibitor cocktail (Roche, Mannheim, Germany), and 2 mM of dithiothreitol. Cell lysates were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were incubated at 4°C with anti-iNOS (1:2,000 dilution) (BD Biosciences, San Jose, CA, USA), anti-phospho-TBK1 (1:1,000 dilution) (Cell Signaling Technology, Beverly, MA, USA), anti-total-TBK1 (1:1,000 dilution) (Cell Signaling Technology, Beverly, MA, USA), and anti‐β‐actin (1:2,000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) for 24 h. After the primary antibody is attached, the relevant secondary antibodies were attached at room temperature for 2 h. Proteins were detected by an enhanced chemiluminescence reagent (iNtRON Biotechnology, Seongnam, Korea).

BMDMs and MH-S cells were seeded on a 48-well plate, 200 μl per well, with a concentration of 1 × 10⁶ cell/ml. After bacterial infection and reagent treatment according to each condition, cell culture supernatants were harvested, and NO concentrations were measured via the Griess reaction assay as previously described (30).

To investigate the difference in inflammatory cytokine production depending on the presence or absence of type I IFN signal in the lung of MAB-infected mice, IL-6 (assay range 15.6–1,000 pg/ml), TNF-α (assay range 31.2–2,000 pg/ml), IL-10 (assay range 31.2–2,000 pg/ml), and IL-1β (assay range 15.6–1,000 pg/ml) cytokines from the supernatant of lung lysate were measured by ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. The cytokine value of the tissue sample was normalized to tissue weight (g).

To elucidate the difference in cytotoxicity depending on the presence or absence of type I IFN signal in the MAB infection, BMDMs and MH-S cells were seeded on a 48-well plate, 200 μl per well, with a concentration of 1 × 10⁶ cell/ml. After bacterial infection and reagent treatment according to each condition, cell culture supernatant was harvested, and lactate dehydrogenase (LDH) levels were measured by CytoTox 96^® Non-Radioactive Cytotoxicity Assay kits (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

For intranasal inoculation or administration, mice were anesthetized with combination of xylazine (10 mg/kg) and zoletil (30 mg/kg). Mice were inoculated with 2 × 10⁷ CFU (in 40 μl of D-PBS) of MAB intranasally. Eight hundred units (in 40 μl of D-PBS) per mouse of rIFN-β was administered in the same way as bacterial inoculation. At indicated day post infection, mice were sacrificed, and lung tissues were harvested in a sterilized condition. The rest of the lobes except the left lobe were homogenized with 500 μl of D-PBS, and the lysate was used for CFU measurements. The remaining lysate was centrifuged for 10 min at 4,000 RCF, and the supernatant was harvested for NO measurement and cytokine analysis. NO concentrations were measured by the Griess reaction assay, and cytokines were analyzed by ELISA. The left lung lobe was used for the histological examination.

Clodronate liposomes and control liposomes were purchased from LIPOSOMA (Amsterdam, Netherlands, Cat# CP-005-005). Animal experiments for the depletion of macrophages in the lungs were performed as follows: 50 μl of clodronate liposomes or control liposomes was administrated intranasally under anesthesia once a day for 3 days. The administration of rIFN-β and bacterial infection were performed according to the schedule indicated in diagram of result section.

The left lobes of the lung tissue were pooled by each group and slightly chopped using a pair of scissors in cold RPMI 1640 containing 1 μg/ml of dipase II (Sigma Aldrich, St. Louis, MO, USA). Samples were incubated at 37°C, 1,100 RPM shaking chamber. After 1 h, samples were placed on 40-μm cell strainer and homogenized using syringe rubber to obtain the single cells. RBCs were lysed using RBC lysis buffer, and flow cytometry assay was performed as previously described (31). In briefly, cells were stained with anti-CD45-APC (BD Biosciences, San Jose, CA, USA), anti-CD11c-PE (BD Biosciences), and anti-F4/80-FITC (Invitrogen, Carlsbad, CA, USA). Analysis was performed by using MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany).

The left lobe of the lung was fixed in 10% neutral formalin for 24 h, followed by tissue processing and paraffin embedding. The paraffin blocks were sectioned at 2 μm and stained with H&E. Histopathological examination was performed under microscopy. Histopathological severity was scored blindly by two experts in a field of laboratory animal pathology with an arbitrary scoring index based on the degree of inflammatory cell infiltration and the extent of the lesion area (0, normal; 1, mild; 2, mild to moderate; 3, moderate; 4, moderate to severe; and 5, severe).

The statistical significance of differences between two groups was determined by unpaired t-test. In case of more than three groups, the statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test for comparisons between groups (GraphPad Prism 5; GraphPad Software Inc., La Jolla, CA, USA). p-Values <0.05 were considered statistically significant.

We first examined whether MAB induces gene expression of type I IFNs in macrophages. Consistent with previous studies (25, 26), MAB upregulated IFN-β gene expression approximately 125-fold compared with 0 h in BMDMs as well as fivefold in MH-S cells, a murine AM cell line, at 3 h after infection ( Figures 1A, B ). Various PRR signaling mediates type I IFN expression by activating TBK1 (5). As expected, infection with MAB induced phosphorylation of TBK1 in both BMDMs and MH-S cells ( Figures 1C, D ). Pretreatment with BX795, a TBK1-specific inhibitor, suppressed MAB-induced TBK1 phosphorylation and IFN-β gene expression in a dose-dependent manner in BMDMs ( Figures 1E, F ), indicating that MAB induces gene expression of type I IFNs in macrophages via TBK1-dependent manner.

There has been an extreme controversy on the role of type I IFNs in the intracellular survival of MAB within macrophages (25, 26). To clarify this, we examined the intracellular bacterial CFUs under various experimental conditions. Compared with WT BMDMs (6.05 log CFU/ml), the bacterial CFUs were higher in IFNAR-deficient BMDMs (6.32 log CFU/ml) at 72 h after infection ( Figure 1G ). In addition, treatment of recombinant IFN-β (rIFN-β) enhanced the bacterial clearance from 6.34 log CFU/ml to 5.66 log CFU/ml in WT BMDMs, but not in IFNAR-deficient cells ( Figures 1H, I ) at 72 h after infection. In MH-S cells, the bacterial clearance was accelerated by rIFN-β from 6 log CFU/ml to 5.55 log CFU/ml at 72 h after infection ( Figure 1J ). These results suggest that type I IFNs inhibit the intracellular growth of MAB in macrophages.

Antimicrobial peptides (AMPs), reactive oxygen species (ROS), and NO are the major factors involved in the removal of intracellular pathogens in macrophages (32). We sought to determine whether type I IFNs regulate expression of those factors in response to MAB in macrophages. IFNAR deficiency did not affect the gene expression of CRAMP and NOX2 ( Figures 2A, B ), whereas iNOS expression was mostly abolished in IFNAR-deficient BMDMs ( Figure 2C ). Treatment with rIFN-β also enhanced MAB-induced iNOS expression in WT BMDMs, but not in IFNAR-deficient BMDMs ( Figure 2D ). Moreover, rIFN-β also increased MAB-induced iNOS expression in MH-S cell ( Figure 2E ). The protein expression of iNOS induced by MAB was reduced in IFNAR-deficient macrophages versus the WT cells ( Figure 2F ), and rIFN-β enhanced MAB-induced expression of iNOS protein in BMDMs ( Figure 2G ) and MH-S cells ( Figure 2H ), but not in IFNAR-deficient BMDMs ( Figure 2G ). Our previous study revealed that MAB alone could not produce detectable level of NO in BMDMs, but at the presence of the type II IFN, IFN-γ, the bacterium led to substantial level of NO production (16). Consistently, at the presence of IFN-γ, MAB induced NO production in WT BMDMs, which were mostly abolished in IFNAR-deficient cells ( Figure 2I ). Unlike BMDMs, MAB alone could produce detectable levels of NO in MH-S cells, which were also enhanced by rIFN-β ( Figure 2J ). These results suggest that type I IFN signaling may contribute to MAB-induced NO production in macrophages.

It has been reported that NO is critical for the restriction of intracellular MAB growth in macrophages (16, 25, 33). As type I IFNs enhanced the intracellular MAB clearance and MAB-induced NO production in BMDMs at the presence of IFN-γ and MH-S cells, we sought to clarify whether NO is a key factor for type I IFN-mediated inhibition of the bacterial growth. In the presence of l-NAME, a non-selective nitric oxide synthase (NOS) inhibitor, there was no significant difference in the bacterial CFUs between WT and IFNAR-deficient BMDMs ( Supplementary Figure 1A ). Treatment of rIFN-β also did not affect the intracellular growth of MAB in l-NAME-treated or iNOS-deficient BMDMs ( Supplementary Figures 1B, C ). As shown in Figure 3A , l-NAME reduced MAB-induced or rIFN-β-enhanced NO production in MH-S cells. Treatment with l-NAME or rIFN-β did not affect the phagocytosis of MH-S cells ( Figure 3B ). Consistent with the result presented in Figure 1J , rIFN-β inhibited the MAB growth 3.8-fold compared with medium in MH-S cells, which was restored by l-NAME treatment ( Figure 3C ). This phenomenon was also confirmed in primary isolated murine AMs. The intracellular growth of MAB was 1.86-fold higher in l-NAME-treated AMs compared with medium-treated AMs at 72 h post infection ( Figure 3D ). Also, while rIFN-β inhibited the growth of MAB 1.91-fold compared with medium control, in the presence of l-NAME, rIFN-β did not suppress the intracellular growth of MAB in AMs, 72 h after infection ( Figure 3D ). These results indicate that type I IFNs suppress the intracellular survival of MAB by promoting NO production in macrophages.

Next, we sought to determine in vivo role of type I IFNs against pulmonary infection of MAB by pre-exposure to rIFN-β. The experimental schedule is depicted in Figure 4A . NO levels in the lung homogenate of mice pretreated (i.n.) with rIFN-β were higher than those of the mice treated with PBS at day 1 after infection, although the difference between the two groups was not observed at day 5 ( Figure 4B ). Pretreatment of rIFN-β also reduced the bacterial CFUs in the lung homogenates of MAB-infected mice at day 5, but not at day 1 ( Figure 4C ). To determine the in vivo effect of type I IFN-induced NO on the growth control of MAB, we repeated the experiment using iNOS-deficient mice. As presented in Figure 4D , intranasal pretreatment with rIFN-β did not influence the MAB growth in the lungs of iNOS-deficient mice ( Figure 4D ), strongly supporting the notion that intranasal pre-exposure to rIFN-β contributes to in vivo growth control of MAB by promoting NO production in macrophages. However, administration of rIFN-β after MAB infection did not improve the bacterial clearance in the lungs of MAB-infected mice ( Supplementary Figures 2A, B ). To determine the impact of type I IFNs on host defense against MAB infection, we compared the bacterial CFUs in the lungs of WT and IFNAR-deficient mice. Unexpectedly, the bacterial CFUs at day 5 were significantly lower in IFNAR-deficient mice as compared with WT mice ( Supplementary Figures 3A, B ), suggesting that type I IFNs can exert a harmful effect in host defense against MAB infection, although prophylactic administration of type I IFNs improves in vivo growth control of MAB.

We investigated whether macrophages are involved in the in vivo protective effect of rIFN-β against the pulmonary infection of MAB. Schedule of clodronate injection and MAB infection is depicted in Figure 5A . The bacterial CFUs in the lungs were examined at day 5 after infection and NO production at day 1. Flow cytometry analysis showed that MAB infection increased the ratio of the CD45⁺F4/80⁺CD11c⁺ AM in the lungs from 4% to 18%, which was rescued to 4% by the treatment with clodronate liposome ( Figure 5A ). In mice treated with clodronate, rIFN-β treatment did not suppress the bacterial growth in the lungs, whereas the bacterial CFUs were decreased by rIFN-β treatment in mice with PBS liposome ( Figure 5B ). Moreover, rIFN-β treatment could not elicit NO production in mice treated with clodronate liposome ( Figure 5C ). Based on these data, it is likely that macrophages are essential for the protective effect of rIFN-β against MAB infection.

Our previous study demonstrated that NOD2 contributes to in vivo and in vitro bacterial clearance against MAB infection by promoting NO production (16). In addition, Pandey et al. reported that NOD2-Ripk2 signaling is involved in induction of type I IFNs against M. tuberculosis infection through bacterial ESX-1- and host TBK1-IRFs-dependent pathways (34). Thus, we hypothesized that NOD2 mediates MAB-induced type I IFN expressions, which results in NO-mediated killing of MAB. NOD2 deficiency diminished MAB-induced phosphorylation of TBK1 in BMDMs ( Figure 6A ). In addition, the gene expression of IFN-β in response to MAB was significantly reduced in NOD2-deficient BMDMs as compared with WT cells ( Figure 6B ). The protein expression of iNOS was also decreased in NOD2-deficient BMDMs, which was restored by the addition of rIFN-β ( Figure 6C ). In an in vivo experiment, MAB-induced NO production was decreased in NOD2-deficient mice at day 1, which was also restored by rIFN-β ( Figure 6D ). The bacterial clearance in the lungs was impaired in NOD2-deficient mice at day 5 ( Figure 6E ). However, intranasal administration of rIFN-β reduced the bacterial CFUs in the lungs of NOD2-deficient mice, exhibiting comparable levels of CFUs as in WT mice ( Figure 6E ). These results suggest that NOD2 signaling contributes to the clearance of MAB by promoting type I IFN-mediated NO production in macrophages.