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

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.

Figure 1

A dose response of SPLUNC1 production in NCI-H292 cells. Cells were cultured in 6-well plates with Mycoplasma pneumoniae (Mp, 1 - 10 cfu/cell) or a TLR2 agonist (Pam3CSK4, 10 - 1000 ng/ml) for 48 hrs, followed by collection of supernatants and cell lysates for SPLUNC1 mRNA and protein measurements, respectively. Data are expressed as means ± SEM (N = 6 replicates). (A) Mp increased SPLUNC1 protein levels in cell supernatants in a dose-dependent manner. (B) Mp increased SPLUNC1 mRNA expression at 1 and 5 cfu/cell. (C) Pam3CSK4 at 100 and 1000 ng/ml significantly augmented SPLUNC1 protein levels in cell supernatants. ND = Not detectable. (D) Pam3CSK4 enhanced SPLUNC1 mRNA expression in a dose-dependent fashion. (E) Western blot analysis of intracellular SPLUNC1 protein and β-actin (a protein loading control) in Mp (10 cfu/cell) and Pam3CSK4 (100 ng/ml)-treated cells using the Odyssey Imaging System (two color detection). Both Mp and Pam3CSK4 increased SPLUNC1 protein.

Figure 2

Effects of Mycoplasma pneumoniae (Mp) and a TLR2 agonist (Pam3CSK4) on SPLUNC1 production in cultured mouse tracheal epithelial cells. Tracheal epithelial cells from TLR2^+/+ and TLR2^-/- mice on the BALB/c background were isolated and cultured under the air-liquid interface (ALI) conditions for 10 days as described in the Methods section. After 48 hrs of Mp (10 cfu/cell) or Pam3CSK4 (1 μg/ml) treatment, SPLUNC1 protein levels in apical supernatants of tracheal epithelial cells were measured using Western blot, quantified using densitometry, and normalized to non-treated (-) cells to obtain SPLUNC1 protein relative levels. As compared to the non-treatment control (-), Mp or Pam3CSK4 treatment in tracheal epithelial cells from TLR2^+/+, but not TLR2^-/- mice, significantly increased SPLUNC1 protein levels. Data are expressed as means ± SEM (N = 3-4 replicates).

Figure 3

TLR2 dependence of SPLUNC1 production in normal human bronchial epithelial cells (NHBE). NHBE (N = 3) were transduced with firefly luciferase short hairpin RNA (Luc shRNA, control) or TLR2 short hairpin RNA (TLR2 shRNA). Thereafter, cells were seeded onto 12-well transwell plates for air-liquid interface (ALI) culture for 10 days, and were then treated with or without Mycoplasma pneumoniae (Mp, 10 cfu/cell) or Pam3CSK4 (1 μg/ml) for 48 hrs. Apical supernatants were collected for SPLUNC1 protein measurement using an ELISA. The paired t test (for normally distributed data under Luc shRNA conditions) or Wilcoxon matched pairs test (for non-parametric data under TLR2 shRNA conditions) was used to analyze the treatment effect of Mp or Pam3CSK4 (Pam3) on SPLUNC1 protein levels. While Mp and Pam3CSK4 increased SPLUNC1 in NHBE transduced with Luc shRNA, they failed to do so in TLR2 shRNA-transduced cells.

Figure 4

Effects NF-κB on SPLUNC1 production in normal human bronchial epithelial cells (NHBE). NHBE were cultured under air-liquid interface (ALI) conditions. At day 10 of ALI culture, cells were treated with an NF-κB p65 inhibitor helenalin (10 μM) for 2 hrs, followed by Mycoplasma pneumoniae (Mp, 10 cfu/cell) infection for 48 hrs. (A) Helenalin inhibited Mp-induced SPLUNC1 protein at the apical surface of NHBE; (B) NF-κB p65 activity was measured in the extracted nuclear proteins of NHBE using an ELISA-based assay (Active Motif, Carlsbad, CA). Mp significantly increased NF-κB p65 activity, which tended (p = 0.07) to be decreased by helenalin. Data are expressed as means ± SEM (n = 3 replicates).

Figure 5

Effects NF-κB pathway on SPLUNC1 production in primary mouse tracheal epithelial cells. Tracheal epithelial cells from CC10-tetracycline-inducible CA-IKKβ (CC10-CA-IKKβ) transgene positive and negative C57BL/6 mice were isolated and cultured under the air-liquid interface conditions for 10 days. Cells were then treated with water (H₂O) or doxycycline (Dox) for 48 hrs. SPLUNC1 mRNA in epithelial cells was quantified by using real-time PCR. As compared to H₂O treatment, Dox significantly increased SPLUNC1 mRNA levels in epithelial cells from CC10-CA-IKKβ transgene positive (Tg+), but not from transgene negative (Tg-) mice. Data are expressed as means ± SEM (N = 4 replicates).

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).