Immune Netw.  2011 Apr;11(2):123-133. 10.4110/in.2011.11.2.123.

Mycobacterial Heparin-binding Hemagglutinin Antigen Activates Inflammatory Responses through PI3-K/Akt, NF-kappaB, and MAPK Pathways

• Kim KH
• Yang CS
• Shin AR
• Jeon SR
• Park JK
• Kim HJ
• Jo EK

• KMID: 2150700
• DOI: http://doi.org/10.4110/in.2011.11.2.123

Abstract

BACKGROUND
Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages.
METHODS
HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-kappaB, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA.
RESULTS
HBHA robustly activated the expression of mRNA and protein of both TNF-alpha and IL-6, and induced phosphorylation of NF-kappaB, Akt, and MAPKs in BMDMs. Both TNF-alpha and IL-6 production by HBHA was regulated by the NF-kappaB, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways.
CONCLUSION
These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.

Keyword

Mycobacterium tuberculosis; Heparin-binding hemagglutinin (HBHA); Macrophages; MAPK; PI3-K; NF-kappaB

MeSH Terms

Cytokines
Enzyme-Linked Immunosorbent Assay
Hemagglutinins
Heparin
Interleukin-6
Lectins
Macrophages
Mycobacterium tuberculosis
NF-kappa B
p38 Mitogen-Activated Protein Kinases
Phosphorylation
RNA, Messenger
Tuberculosis
Tumor Necrosis Factor-alpha
Cytokines
Hemagglutinins
Heparin
Interleukin-6
Lectins
NF-kappa B
RNA, Messenger
Tumor Necrosis Factor-alpha
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 Kinetics of proinflammatory responses induced by HBHA in macrophages. BMDMs were stimulated with HBHA (1µg/ml) for the indicated periods of time. (A) Cells were harvested and semi-quantitative RT-PCR analysis of TNF-α and IL-6 mRNA level was performed. The relative densities of the expression levels were analyzed by densitometry. All densitometry values were normalized to that of β-actin mRNA. Top: representative gel images showing the products of RT-PCR analysis, Bottom: densitometric analysis. (B) The supernatants were harvested after the times indicated for assessment of TNF-α and IL-6 levels by ELISA. Mean results and densitometry values are depicted as mean±SD of three independent determinations. Significant differences (*p<0.05, **p<0.01, ***p<0.001) as compared with control cultures.

• 

  Figure 2 Kinetics of NF-κB and MAPK signaling pathway activation in macrophages after stimulation with HBHA. (A) BMDMs were stimulated with HBHA (1µg/ml) for the indicated periods of time. Cell lysates were harvested and subjected to Western blot analysis for phosphorylated IKK-α/β. The same blots were washed and blotted for IκB-α and phosphorylated MAPKs (p-ERK, p-p38, p-JNK). β-actin was probed as a loading control. Data are representative of three independent determinations with similar results. (B) Densitometric analysis of Western blot bands for phospho-IKK-α/β and IκB-α; both were normalized with β-actin. (C) Densitometric analysis of Western blot bands for phospho-ERK, p38, and JNK were normalized to β-actin.

• 

  Figure 3 NF-κB activation is required for HBHA-induced TNF-α and IL-6 production by macrophages. (A) NF-κB translocation by HBHA. BMDMs were stimulated with HBHA (1µg/ml) for 1 h and immunostained with anti-NF-κB-p65 and anti-rabbit-Alexa Fluor 488. Left, representative images of three independent determinations with similar results. Scale bar=20µm. Right: quantification of data, NF-κ B-nuclear translocated cells were counted manually in DAPI-stained BMDMs. Data shown represent the means±SD of three independent samples, with each experiment including at least 200 cells scored in five random fields. (B) BMDMs were preincubated for 45 min with BAY 11-7082 (BAY; 0.3, 1, 3µM), prior to stimulation with HBHA (1µg/ml). Supernatants were harvested at 18 h and protein levels were determined by ELISA. Data represent the mean±SD of three independent determinations. Significant differences (*p<0.05, **p<0.01) as compared with HBHA-treated cells. U:unstimulated, SC:solvent control (0.1% DMSO).

• 

  Figure 4 Effects of the PI3-K/Akt signaling pathways on HBHA-induced TNF-α and IL-6 expression by macrophages. (A) BMDMs were stimulated with HBHA (1µg/ml) for the times indicated. The cell lysates were then harvested and subjected to Western blot analysis for phosphorylated Akt. The same blots were washed and blotted for total Akt as the loading control. Top: representative gel image was shown, Bottom: densitometric analyses of Western blot bands for phospho-Akt was normalized to total Akt. Data are representative of three independent determinations with similar results. (B, C) Cells were pretreated with LY294002 (LY; 5, 10, 20µM) or Wortmannin (WM; 100, 200, 300 nM) for 45 min prior to stimulation with HBHA (1µg/ml) for 6 h (for B) or 18 h (for C). (B) Total RNA was purified and semi-quantitative RT-PCR was used to determine TNF-α and IL-6 expression in BMDMs. Top: representative gel image of three independent replicates was shown, Bottom: the relative densities of expression levels were analyzed by densitometry. All densitometry values were normalized to those of β-actin mRNA. (C) Supernatants were harvested at 18 h, and TNF-α and IL-6 levels were measured by ELISA. Data shown are the mean±SD of three determinations. (D) RAW264.7 cells were transfected with PI3-K p110α-DN or empty vector for 24 h prior to stimulation with HBHA (1µg/ml) for 18 h. The supernatants were then harvested and subjected to ELISA. Significant differences (*p<0.05, **p<0.01, ***p<0.001), compared with HBHA-treated cells or Mock control. U:unstimulated, SC:solvent control (0.1% DMSO).

• 

  Figure 5 MAPK signaling pathways are required for HBHA-induced TNF-α and IL-6 expression by macrophages. (A) BMDMs were pretreated with p38 inhibitor (SB203580; 1, 5, 10µM), MEK-1 inhibitor (U0126; 5, 10, 20µM), or JNK inhibitor (SP600125; 5, 20, 30µM) prior to stimulation with HBHA (1µg/ml). Cells were then harvested at 6 h, and total RNA purified and subjected to semi-quantitative RT-PCR analysis for assessment of TNF-α and IL-6 expression. Top: representative gel image of three independent replicates was shown, Bottom: the relative densities of expression levels were analyzed by densitometry. All densitometry values were normalized to those of β-actin mRNA. (B) BMDMs were pretreated with p38 inhibitor (SB203580, 10µM), MEK-1 inhibitor (PD98059 and U0126, 20µM), or JNK inhibitor (SP600125, 20µM) prior to stimulation with HBHA (1µg/ml). The supernatants from BMDMs were collected 18 h after stimulation of HBHA, and subjected to ELISA analysis for assessment of TNF-α and IL-6 production. Data shown are the mean±SD of three determinations. (C) RAW264.7 cells were transfected with p38 MAPK-DN, MEK-1-DN, or empty vector prior to stimulation with HBHA (1µg/ml) for 18 h. The supernatants from cells were harvested and subjected to ELISA for determination of TNF-α and IL-6 production. Significant differences (*p<0.05, **p<0.01, ***p<0.001) as compared with control cultures. U:unstimulated, SC:solvent control (0.1% DMSO), SB:SB203580, PD:PD98059, SP:SP600125.

• 

  Figure 6 PI3-K/Akt pathway is the upstream signaling activator for p38 and ERK1/2 signaling pathways in macrophages after stimulation with HBHA. (A) BMDMs were incubated with LY294002 (LY; 5, 10, 20µM) or Wortmannin (WM; 100, 200, 300 nM) for 45 min prior to treatment with HBHA (1µg/ml). Cell lysates were then harvested (for p-Akt, at 5 min; for MAPKs, at 30 min), and subjected to Western blot analysis for p-Akt and MAPKs (p38, ERK, JNK). The same blots were stripped, washed, and re-probed with β-actin as a loading control. Data are representative of three independent determinations with similar results. (B) Densitometric analysis of the Western blot bands for p-Akt, p-ERK, p-p38, and p-JNK were normalized to total-Akt (for p-Akt) or β-actin (for MAPKs). Significant differences (*p<0.05, **p<0.01, ***p<0.001), compared with control cultures. U:unstimulated, SC:solvent control (0.1% DMSO).

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