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SPLUNC1 regulation in airway epithelial cells: role of Toll-like receptor 2 signaling.

Chu HW, Gally F, Thaikoottathil J, Janssen-Heininger YM, Wu Q, Zhang G, Reisdorph N, Case S, Minor M, Smith S, Jiang D, Michels N, Simon G, Martin RJ - Respir. Res. (2010)

Bottom Line: Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases.However, the mechanisms underlying SPLUNC1 regulation remain unknown.Our data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, National Jewish Health, and the University of Colorado Denver, Denver, CO, USA. chuhw@njhealth.org

ABSTRACT

Background: Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases. We have shown that mouse short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein was able to inhibit Mp growth. Further, airway epithelial cells increased SPLUNC1 expression upon Mp infection. However, the mechanisms underlying SPLUNC1 regulation remain unknown. In the current study, we investigated if SPLUNC1 production following Mp infection is regulated through Toll-like receptor 2 (TLR2) signaling.

Methods: Airway epithelial cell cultures were utilized to reveal the contribution of TLR2 signaling including NF-κB to SPLUNC1 production upon bacterial infection and TLR2 agonist stimulation.

Results: Mp and TLR2 agonist Pam3CSK4 increased SPLUNC1 expression in tracheal epithelial cells from wild type, but not TLR2(-/-) BALB/c mice. RNA interference (short-hairpin RNA) of TLR2 in normal human bronchial epithelial cells under air-liquid interface cultures significantly reduced SPLUNC1 levels in Mp-infected or Pam3CSK4-treated cells. Inhibition and activation of NF-κB pathway decreased and increased SPLUNC1 production in airway epithelial cells, respectively.

Conclusions: Our data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

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Purity, specificity and antimicrobial activity of recombinant human SPLUNC1 protein. (A) Left-panel - Two μg of SPLUNC1 protein was electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred onto a nitrocellulose membrane. Ponceau S staining of the membrane showed one protein at about 25 kD (pink, black arrow). Right panel - Western blot of SPLUNC1 using the Odyssey Imaging System verified that the 25 kD protein band shown in the left panel was SPLUNC1 (green band). (B) A representative fragmentation spectrum of peptide LYVTIPLGIK (amino acids 129 to 138) from in-gel trypsin-digested recombinant hSPLUNC1 protein after matching algorithm using SpectrumMill. The y-axis indicates the relative intensity of the fragment ions, where 100% is the total ion intensity of the spectrum. (C) Recombinant human SPLUNC1 protein markedly reduced Mycoplasma pneumoniae (Mp) growth in 96-well culture plates for 2 hrs. CFUs = Colony forming units. Data are expressed as means ± SEM (N = 5 replicates).
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Figure 6: Purity, specificity and antimicrobial activity of recombinant human SPLUNC1 protein. (A) Left-panel - Two μg of SPLUNC1 protein was electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred onto a nitrocellulose membrane. Ponceau S staining of the membrane showed one protein at about 25 kD (pink, black arrow). Right panel - Western blot of SPLUNC1 using the Odyssey Imaging System verified that the 25 kD protein band shown in the left panel was SPLUNC1 (green band). (B) A representative fragmentation spectrum of peptide LYVTIPLGIK (amino acids 129 to 138) from in-gel trypsin-digested recombinant hSPLUNC1 protein after matching algorithm using SpectrumMill. The y-axis indicates the relative intensity of the fragment ions, where 100% is the total ion intensity of the spectrum. (C) Recombinant human SPLUNC1 protein markedly reduced Mycoplasma pneumoniae (Mp) growth in 96-well culture plates for 2 hrs. CFUs = Colony forming units. Data are expressed as means ± SEM (N = 5 replicates).

Mentions: Our recent publication demonstrated that recombinant mouse SPLUNC1 protein significantly inhibited the growth of Mp in a dose-dependent manner [7]. To verify if human SPLUNC1 protein exerts a similar activity to the mouse counterpart in inhibiting Mp growth, we generated recombinant human SPLUNC1 (hSPLUNC1) protein using a baculovirus expression system. The purity and specificity of hSPLUNC1 protein were verified by Western blot and mass spectrometry (Figure 6A and 6B). As shown in Figure 6C, hSPLUNC1 inhibited Mp growth in a dose-dependent manner.


SPLUNC1 regulation in airway epithelial cells: role of Toll-like receptor 2 signaling.

Chu HW, Gally F, Thaikoottathil J, Janssen-Heininger YM, Wu Q, Zhang G, Reisdorph N, Case S, Minor M, Smith S, Jiang D, Michels N, Simon G, Martin RJ - Respir. Res. (2010)

Purity, specificity and antimicrobial activity of recombinant human SPLUNC1 protein. (A) Left-panel - Two μg of SPLUNC1 protein was electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred onto a nitrocellulose membrane. Ponceau S staining of the membrane showed one protein at about 25 kD (pink, black arrow). Right panel - Western blot of SPLUNC1 using the Odyssey Imaging System verified that the 25 kD protein band shown in the left panel was SPLUNC1 (green band). (B) A representative fragmentation spectrum of peptide LYVTIPLGIK (amino acids 129 to 138) from in-gel trypsin-digested recombinant hSPLUNC1 protein after matching algorithm using SpectrumMill. The y-axis indicates the relative intensity of the fragment ions, where 100% is the total ion intensity of the spectrum. (C) Recombinant human SPLUNC1 protein markedly reduced Mycoplasma pneumoniae (Mp) growth in 96-well culture plates for 2 hrs. CFUs = Colony forming units. Data are expressed as means ± SEM (N = 5 replicates).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992501&req=5

Figure 6: Purity, specificity and antimicrobial activity of recombinant human SPLUNC1 protein. (A) Left-panel - Two μg of SPLUNC1 protein was electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred onto a nitrocellulose membrane. Ponceau S staining of the membrane showed one protein at about 25 kD (pink, black arrow). Right panel - Western blot of SPLUNC1 using the Odyssey Imaging System verified that the 25 kD protein band shown in the left panel was SPLUNC1 (green band). (B) A representative fragmentation spectrum of peptide LYVTIPLGIK (amino acids 129 to 138) from in-gel trypsin-digested recombinant hSPLUNC1 protein after matching algorithm using SpectrumMill. The y-axis indicates the relative intensity of the fragment ions, where 100% is the total ion intensity of the spectrum. (C) Recombinant human SPLUNC1 protein markedly reduced Mycoplasma pneumoniae (Mp) growth in 96-well culture plates for 2 hrs. CFUs = Colony forming units. Data are expressed as means ± SEM (N = 5 replicates).
Mentions: Our recent publication demonstrated that recombinant mouse SPLUNC1 protein significantly inhibited the growth of Mp in a dose-dependent manner [7]. To verify if human SPLUNC1 protein exerts a similar activity to the mouse counterpart in inhibiting Mp growth, we generated recombinant human SPLUNC1 (hSPLUNC1) protein using a baculovirus expression system. The purity and specificity of hSPLUNC1 protein were verified by Western blot and mass spectrometry (Figure 6A and 6B). As shown in Figure 6C, hSPLUNC1 inhibited Mp growth in a dose-dependent manner.

Bottom Line: Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases.However, the mechanisms underlying SPLUNC1 regulation remain unknown.Our data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, National Jewish Health, and the University of Colorado Denver, Denver, CO, USA. chuhw@njhealth.org

ABSTRACT

Background: Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases. We have shown that mouse short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein was able to inhibit Mp growth. Further, airway epithelial cells increased SPLUNC1 expression upon Mp infection. However, the mechanisms underlying SPLUNC1 regulation remain unknown. In the current study, we investigated if SPLUNC1 production following Mp infection is regulated through Toll-like receptor 2 (TLR2) signaling.

Methods: Airway epithelial cell cultures were utilized to reveal the contribution of TLR2 signaling including NF-κB to SPLUNC1 production upon bacterial infection and TLR2 agonist stimulation.

Results: Mp and TLR2 agonist Pam3CSK4 increased SPLUNC1 expression in tracheal epithelial cells from wild type, but not TLR2(-/-) BALB/c mice. RNA interference (short-hairpin RNA) of TLR2 in normal human bronchial epithelial cells under air-liquid interface cultures significantly reduced SPLUNC1 levels in Mp-infected or Pam3CSK4-treated cells. Inhibition and activation of NF-κB pathway decreased and increased SPLUNC1 production in airway epithelial cells, respectively.

Conclusions: Our data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

Show MeSH
Related in: MedlinePlus