Treatment with the hyaluronic acid synthesis inhibitor 4-methylumbelliferone suppresses SEB-induced lung inflammation

Abstract

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for SEB-induced inflammation. In the current study we investigated the potential use of the hyaluronic acid synthase inhibitor 4-methylumbelliferone (4-MU) on staphylococcal enterotoxin B (SEB) induced acute lung inflammation. Culturing SEB-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production as well as an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from SEB-induced lung injury. Specifically, 4-MU treatment led to a reduction in SEB-induced HA levels, reduction in lung permeability, and reduced pro-inflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target hyaluronic acid production may be an effective treatment for the inflammatory response following exposure to SEB.

Figure 1

4-MU inhibits SEB-induced leukocyte proliferation and cytokine production in vitro. Spleen cells from C57BL/6 mice were treated with 4-MU (0.1, 0.5, and 1.0 mM) or vehicle control (DMSO) and then stimulated with SEB (2 μg/mL). The effect of 4-MU on the proliferative response and cytokine production was determined 48 h later by MTT assay (A) and cytometric bead array (B), respectively. Asterisks indicate statistically significant difference when compared with vehicle controls, p ≤ 0.05.

Figure 2

4-MU treatment leads to increased apoptosis in SEB-exposed leukocytes in vitro. Spleen cells from C57BL/6 mice were treated with 4-MU (0.1, and 0.5 mM) or vehicle control (DMSO) and then stimulated with SEB (2 μg/mL). The effect of 4-MU on SEB-induced apoptosis was determined 48 h later by Annexin V/PI (A) and TUNEL (B) assays, respectively. The level of apoptosis in individual immune cell subsets was determined by staining the spleen cells with phenotype-specific fluorescently-labeled mAbs followed by TUNEL staining (C).

Figure 3

4-MU treatment suppresses SEB-induced hyaluronic acid synthase expression and accumulation of soluble HA in the lungs. The effect of 4-MU on soluble HA levels in the lungs of SEB-exposed mice was determined by treating the mice with 4-MU (450 mg/mouse i.p.) or vehicle control (5% gum arabic) one day prior and on the day of SEB exposure (20 µg/50 μL PBS). The levels of lung hyaluronic acid and mRNA levels of HAS were determined 24 h later. HAS mRNA levels in whole lung extracts were determined by real-time RT-PCR (A). Hyaluronic acid levels in bronchoalveolar lavage fluid (BALF) were determined by ELISA (B). Asterisks indicate statistically significant difference when compared with the levels from vehicle-exposed mice, p ≤ 0.05. Number sign indicates statistically significant difference when compared to PBS exposed mice, p ≤ 0.05.

Figure 4

The effect of 4-MU on SEB-induced vascular permeability in vivo was determined by exposing mice to SEB, as described in the Material and Methods section, and treating the mice with 4-MU (450 mg/mouse i.p.) or Vehicle (5% gum arabic i.p.) one day prior, and on the day of, SEB exposure. Vascular permeability was determined as described Materials and Methods. Asterisks indicate statistically significant difference when compared with the Vehicle-treated controls, p ≤ 0.05.

Figure 5

4-MU treatment suppresses SEB-induced inflammatory cytokine production in the lungs. The effect of 4-MU on SEB-induced inflammatory cytokine production in vivo was determined by treating the mice with 4-MU (450 mg/mouse i.p.) or vehicle (300 µL 5% gum arabic/mouse i.p.) one day prior to, and on the day of, SEB exposure. Following 4-MU treatment mice were exposed to SEB or PBS, as described in the Material and Methods section. The levels of BALF cytokine protein levels and total lung cytokine mRNA and were determined 24 h later. Cytokine mRNA levels in whole lung extracts were determined by real-time RT-PCR (A). The protein levels of cytokines in BALF were determined using a cytokine bead array (B). Asterisks indicate statistically significant difference when compared to the cytokine levels from SEB-exposed vehicle-treated mice, p < 0.05.