Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation

Abstract

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

T cell-directed IL-17 production by lung granular γδ T cells is coordinated by a novel IL-2 and IL-1β circuit

“…BAL was centrifuged at 1000 rpm at 4 °C to separate cells from BALF and 0.2 μm filtered as described earlier. 28 Lungs were homogenized post-BAL lavage using MagNA Lyser Green Beads (Roche, Basel, Switzerland) at 6000 rpm. Protein was quantified using BCA assay (Thermo Fisher Scientific, Waltham, MA).…”

CD169+ Macrophages Restrain Systemic Inflammation Induced by Staphylococcus aureus Enterotoxin A Lung Response

“…Similarly, immunotherapy using Abs specific to superantigens was found to be protective in animal models (42). In addition to Abs, it has been shown that lactoferrin, an iron-binding glycoprotein located predominantly in mucosal secretions (43), can attenuate the inflammatory responses triggered by S. aureus enterotoxins (35). In this study we show that AMs may represent a key cellular defense mechanism that can reduce the effects of S. aureus enterotoxin-induced inflammation.…”

“…Furthermore, using a titration curve, the concentration of enterotoxin A in serum after inhalation was estimated to be only a small fraction of the total amount inhaled by a mouse (;,1/35th; J. Svedova, unpublished observations). Intriguingly, in another report using mass spectrometry, we showed that enterotoxin A could be recovered from BAL fluid at 16 h after inhalation and the toxin preserved its biological activity (35). These findings suggested that when enterotoxin A is inhaled, only a small portion enters the circulation, whereas the remainder may be retained in the lung mucosa or perhaps neutralized by various defense mechanisms, such as defensins or phagocytosis.…”