Type I Interferons Are Involved in the Intracellular Growth Control of Mycobacterium abscessus by Mediating NOD2-Induced Production of Nitric Oxide in Macrophages

Abstract

Mycobacterium abscessus (MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-β treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/β receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-β treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-β before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-β did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-β gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-β treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.

Keywords: Mycobacterium abscessus; NOD2; macrophage; nitric oxide; type I IFN.

Figure 1

MAB induces IFN-β gene expression of macrophages in TBK1-dependent manner, which promotes intracellular bacterial clearance. (A, B) BMDMs and mouse alveolar macrophage cell line MH-S cells were infected with MAB at multiplicity of infection (MOI) 1:25 for indicated times. mRNA was extracted, and the expression levels of IFN-β gene were determined by real-time PCR. (C) BMDMs and (D) MH-S cells were infected with MAB for indicated times. (E) BMDMs were infected with MAB for 1 h in the absence or presence of BX795 (2 h pretreated). (C–E) The levels of indicated proteins were determined by Western blotting. (F) BMDMs were infected with MAB for 6 h in the absence or presence of BX795 (2 h pretreated). The mRNA was extracted, and the expression levels of IFN-β gene were determined by real-time PCR. (G–J) Cells were infected with MAB in the absence or presence of recombinant IFN-β (1,000 units/ml, 2 h pretreated). Intracellular bacterial CFU on indicated times were evaluated by intracellular bacterial growth assay. (A–J) The results are from one representative experiment of two independent experiments (**p < 0.01, ***p < 0.001). MAB, Mycobacterium abscessus; IFN, interferon; TBK1, TANK binding kinase 1; BMDMs, bone marrow-derived macrophages; CFU, colony-forming unit. NS, Not Statistically Significant.

Figure 2

Type I IFNs augment MAB-induced production of nitric oxide in macrophages. (A–C) WT and IFNAR-deficient BMDMs were infected with MAB at a MOI 1:25 for indicated times. (D, E) BMDMs and MH-S cells were pretreated with or without rIFN-β (1,000 units/ml) for 2 h and additionally infected with MAB for (D) 12 h or (E) indicated times. (A–E) mRNA was extracted, and the expression levels of each gene were determined by real-time PCR. (F) BMDMs were infected with MAB for indicated times. (G) BMDMs and (H, J) MH-S cells were pretreated with or without rIFN-β (1,000 units/ml) for 2 h and additionally infected with MAB for (G, H) 24 h or (J) indicated times. (I) BMDMs were incubated for 24 h with indicated conditions. (F–H) Cellular proteins were extracted, and the levels of indicated proteins were determined by Western blotting. (I, J) Nitric oxide concentration in cell culture supernatant was measured by Griess reaction. (A–J) The results are from one representative experiment of two independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). MAB, Mycobacterium abscessus; IFN, interferon; WT, wild type; IFNAR, interferon-α/β receptor; BMDMs, bone marrow-derived macrophages; MOI, multiplicity of infection. NS, Not Statistically Significant.

Figure 3

Type I IFNs inhibits the intracellular MAB growth in macrophages by regulating NO production. (A–C) MH-S cells were incubated with MAB and indicated reagents (2 h pretreated). (A) Nitric oxide concentration in cell culture supernatant was measured by Griess reaction at 24 h post infection. Intracellular bacterial CFU on (B) 1 h and (C) 72 h were evaluated by intracellular bacterial growth assay. (D) AMs were incubated with MAB (MOI 1:25) and indicated reagents (2 h pretreated, l-NAME 1 mM, rIFN-β 1,000 units/ml). Intracellular bacterial CFU on 72 h was evaluated by intracellular bacterial growth assay. (A–D) The results are from one representative experiment of two independent experiments (**p < 0.01, ***p < 0.001). MAB, Mycobacterium abscessus; IFN, interferon; CFU, colony-forming unit; AMs, alveolar macrophages; MOI, multiplicity of infection. NS, Not Statistically Significant.

Figure 4

Intranasal pretreatment of rIFN-β augments NO production and promotes the bacterial clearance in the lungs of mice infected with MAB. (A) The administration of rIFN-β and bacterial infection were performed according to the schedule indicated in the diagram. (B–D) WT or iNOS-deficient mice were administrated with PBS or rIFN-β (800 units per mouse) intranasally under anesthesia. After 1 day, mice were infected with 2 × 10⁷ CFU of MAB per mouse intranasally; and the bacterial loads and nitric oxide levels in the lung lysate were determined at indicated days. (B–D) The results are merged data of two independent experiments (n = 4–6) (***p < 0.001). MAB, Mycobacterium abscessus; WT, wild type; PBS, phosphate-buffered saline; CFU, colony-forming unit. NS, Not Statistically Significant.

Figure 5

Macrophages are responsible for rIFN-β response in MAB-induced NO production and the bacterial clearance in vivo. (A) The administration of reagents and bacterial infection were performed according to the schedule indicated in the diagram. Flow cytometric plot showing the population of CD45⁺CD11c⁺F4/80⁺ alveolar macrophage in lung total cell at 1 day post infection. (B) Mice were administrated with indicated reagents intranasally under anesthesia. After 1 day, mice were infected with 2 × 10⁷ CFU of MAB per mouse intranasally, and the bacterial loads in the lung lysate were determined at 5 days post infection. (C) Nitric oxide levels were measured on 1 day post infection in the lung lysate supernatant. (A) The results are one representative data of two independent experiments (n = 4). (B, C) The results are merged data of two independent experiments (n = 4–5) (**p < 0.01, ***p < 0.001). MAB, Mycobacterium abscessus; CFU, colony-forming unit. NS, Not Statistically Significant.

Figure 6

Type I IFNs act as an intermediator of a cytosolic receptor NOD2-mediated NO production in response to MAB. (A) BMDMs were infected with MAB for indicated times. Cellular proteins were extracted, and the levels of each protein were determined by Western blotting. (B) BMDMs were infected with MAB for indicated time. mRNA was extracted, and the expression levels of IFN-β gene were determined by real-time PCR. (C) BMDMs were incubated for 24 h with indicated conditions. The levels of each protein were determined by Western blotting. (D) Nitric oxide levels were measured on 1 day post infection in the lung lysate (same conditions with Figure 4 ). (E) The bacterial load in the lung lysate was determined at 5 days post infection. (A–C) The results are from one representative experiment of two independent experiments. (D, E) The results are merged data of two independent experiments (n = 5) (**p < 0.01, ***p < 0.001). MAB, Mycobacterium abscessus; IFN, interferon; BMDMs, bone marrow-derived macrophages.