Immune Netw.  2014 Dec;14(6):307-320. 10.4110/in.2014.14.6.307.

Characterization of Proinflammatory Responses and Innate Signaling Activation in Macrophages Infected with Mycobacterium scrofulaceum

• Kim KH
• Kim TS
• Lee JG
• Park JK
• Yang M
• Kim JM
• Jo EK
• Yuk JM

• KMID: 2150821
• DOI: http://doi.org/10.4110/in.2014.14.6.307

Abstract

Mycobacterium scrofulaceum is an environmental and slow-growing atypical mycobacterium. Emerging evidence suggests that M. scrofulaceum infection is associated with cervical lymphadenitis in children and pulmonary or systemic infections in immunocompromised adults. However, the nature of host innate immune responses to M. scrofulaceum remains unclear. In this study, we examined the innate immune responses in murine bone marrow-derived macrophages (BMDMs) infected with different M. scrofulaceum strains including ATCC type strains and two clinically isolated strains (rough and smooth types). All three strains resulted in the production of proinflammatory cytokines in BMDMs mediated through toll-like receptor-2 and the adaptor MyD88. Activation of MAPKs (extracellular signal-regulated kinase 1/2, and p38, and c-Jun N-terminal kinase) and nuclear receptor (NF)-kappaB together with intracellular reactive oxygen species generation were required for the expression of proinflammatory cytokines in BMDMs. In addition, the rough morphotypes of M. scrofulaceum clinical strains induced higher levels of proinflammatory cytokines, MAPK and NF-kappaB activation, and ROS production than other strains. When mice were infected with different M. scrofulaceum strains, those infected with the rough strain showed the greatest hepatosplenomegaly, granulomatous lesions, and immune cell infiltration in the lungs. Notably, the bacterial load was higher in mice infected with rough colonies than in mice infected with ATCC or smooth strains. Collectively, these data indicate that rough M. scrofulaceum induces higher inflammatory responses and virulence than ATCC or smooth strains.

Keyword

Mycobacterium scrofulaceum; Proinflammatory cytokines; MAPKs; TLR; MyD88; Nuclear receptor-kappaB

MeSH Terms

Adult
Animals
Bacterial Load
Child
Cytokines
Humans
Immunity, Innate
Lung
Lymphadenitis
Macrophages*
Mice
Mycobacterium scrofulaceum*
NF-kappa B
Nontuberculous Mycobacteria
Phosphotransferases
Reactive Oxygen Species
Virulence
Cytokines
NF-kappa B
Phosphotransferases
Reactive Oxygen Species

Figure

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  Figure 1 Mycobacterium scrofulaceum-114R elicits cord formation and enhanced expression of proinflammatory cytokines in BMDMs. (A) The colony type of M. scrofulaceum-ATCC, -113S, and -114R on 7H10 agar plates was characterized microscopically at ×40 (a) and ×100 magnification (b), and the cords formed in broth culture after 14 days of incubation were heat fixed and then stained with crystal violet (c). (B and C) BMDMs were infected with different clinical M. scrofulaceum strains (MOI=3) for the indicated times. The mRNA expression and production of TNF-α, IL-6, and IL-12p40 were evaluated by semi-quantitative RT-PCR (B) and ELISA (C), respectively. The relative expression levels were normalized against those of β-actin (B, bottom). The data show the means±SD from three independent experiments. U; uninfected control.

• 

  Figure 2 Mycobacterium scrofulaceum-induced proinflammatory cytokine production is mediated by TLR2/MyD88 signaling. (A and B) BMDMs isolated from WT and TLR2 KO mice (for A) or WT, MyD88, and TRIF KO mice (for B) were stimulated with M. scrofulaceum-ATCC, -113S, and -114R (MOI=1, 5, 10) for 18 h. The levels of TNF-α, IL-6, and IL-12p40 in the supernatants were measured by ELISA. Pam3CSK4 (P; 100 ng/ml) was used as positive ligand for TLR2. The data represent the mean±SD from three independent experiments. U; uninfected control.

• 

  Figure 3 Mycobacterium scrofulaceum-114R strongly increases the production of proinflammatory cytokines through NF-κB signaling in BMDMs. (A) BMDMs were transduced with NF-κB p65 adenovirus luciferase construct (20 PFU/cells) for 36 h and then infected with M. scrofulaceum-ATCC, -113S, -114R (MOI=1, 5, 10) for 4 h. Cell lysates were harvested and luciferase activity was measured. (B) BMDMs were infected with M. scrofulaceum strains (MOI=5) for 30 min and then immunolabeled with anti-NF-κB p65 antibody and anti-rabbit-AlexaFluor 488 (green), and the nuclei were stained with DAPI (blue). Representative immunofluorescence images (left panel) and the average mean fluorescence intensity of cells exhibiting NF-κB nuclear translocation (right panel) are shown. (C) BMDMs were preincubated in the presence or absence of BAY11-7082 (BAY; 0.3, 1, 3µM) or CAPE (1, 5, 10µM) for 45 min prior to infection with M. scrofulaceum strains (MOI=5). The mRNA expression and production of TNF-α and IL-6 were evaluated by semi-quantitative RT-PCR. The data show the mean±SD from three independent experiments. U; uninfected control, SC; solvent control (0.1% DMSO).

• 

  Figure 4 Mycobacterium scrofulaceum 114R-induced pro-inflammatory cytokine production is modulated through MAPK signaling in BMDMs. (A) Kinetics of phospho-RK1/2, -p38, and -SAPK/JNK in BMDMs infected with M. scrofulaceum-ATCC, -113S, and -114R (MOI=5). The cell lysates were collected at the indicated times and phosphorylated MAPKs were examined by western blot analysis. β-ctin was used as a loading control. (B) Expression levels were normalized against those of β-actin (C) BMDMs were pretreated with p38 inhibitor (SB203580; 1, 5, and 10µM), MEK-1 inhibitor (U0126; 5, 10, and 20µM), and JNK inhibitor (JNK; 5, 10, and 20µM) for 45 min prior to infection with different M. scrofulaceum strains (MOI=5). The culture supernatants were harvested at 18 h, and the production of TNF-α , IL-6, and IL-12p40 cytokines was measured by ELISA. The data show the mean±SD of three independent experiments. Significant differences: #p<0.05 compared with uninfected control; *p<0.05, **p<0.01, and ***p<0.001 compared with solvent control. U; uninfected control, SC; solvent control (0.1% DMSO).

• 

  Figure 5 ROS signaling-mediated proinflammatory cytokine production in Mycobacterium scrofulaceum-infected BMDMs (A) BMDMs were infected with M. scrofulaceum-ATCC, -113S, or -114R (MOI 5, 10) for 30 min and then stained with DHE for 30 min. Superoxideroducing cells were analyzed by flow cytometry. (B) BMDMs were incubated with NAC (10, 20, and 30 mM) or DPI (1, 5, and 10µM) before infection with different M. scrofulaceum strains (MOI=5). The supernatant was harvested at 20 h, and cytokine production was measured by ELISA. The data show the mean±SD of three independent experiments. Significant differences: **p<0.01 and ***p<0.001 compared with solvent control. U; uninfected control, SC; solvent control (0.1% DMSO).

• 

  Figure 6 Mycobacterium scrofulaceum-114R infection induces granulomatous lesion formation, higher mycobacteria burden and inflammation in the lung C57BL/6 mice were infected intravenously with 5×107 CFU of M. scrofulaceum-ATCC, -113S, or 114R or treated with saline (UI) for the indicated periods of time. (A) Lung tissues were collected and fixed with 10% formalin and paraffin-embedded. Lung sections were stained with hematoxylin and eosin (H&E). Inflammatory cell infiltration, granulomatous lesions, and lung histology were observed at ×100 (upper) or ×200 (bottom) magnification; the black arrowhead in the panel indicates the granulomatous lesion. (B) mRNA expression of TNF-α, IL-6, and IL-12p40 cytokines in the lungs was determined by real-time PCR analysis. (C) Expression of COX-2 in lungs of mice infected with M. scrofulaceum strains was examined at 14 days post-infection by immunohistochemical staining of COX-2. (D) The mycobacterial loads of the lungs were determined at 0 and 7 days post-infection. The data show the mean±SD of three individual mice per group. Significant difference: *p<0.05, compared to M. scrofulaceum-ATCC or -113S-infected mice; **p<0.01 compared with the uninfected control (UI).

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