Cytochrome b5 and Cytokeratin 17 Are Biomarkers in Bronchoalveolar Fluid Signifying Onset of Acute Lung Injury

Acute lung injury (ALI) is characterized by pulmonary edema and acute inflammation leading to pulmonary dysfunction and potentially death. Early medical intervention may ameliorate the severity of ALI, but unfortunately, there are no reliable biomarkers for early diagnosis. We screened for biomarkers in a mouse model of ALI. In this model, inhalation of S. aureus enterotoxin A causes increased capillary permeability, cell damage, and increase protein and cytokine concentration in the lungs. We set out to find predictive biomarkers of ALI in bronchoalveolar lavage (BAL) fluid before the onset of clinical manifestations. A cutting edge proteomic approach was used to compare BAL fluid harvested 16 h post S. aureus enterotoxin A inhalation versus BAL fluid from vehicle alone treated mice. The proteomic PF 2D platform permitted comparative analysis of proteomic maps and mass spectrometry identified cytochrome b5 and cytokeratin 17 in BAL fluid of mice challenged with S. aureus enterotoxin A. Validation of cytochrome b5 showed tropic expression in epithelial cells of the bronchioles. Importantly, S. aureus enterotoxin A inhalation significantly decreased cytochrome b5 during the onset of lung injury. Validation of cytokeratin 17 showed ubiquitous expression in lung tissue and increased presence in BAL fluid after S. aureus enterotoxin A inhalation. Therefore, these new biomarkers may be predictive of ALI onset in patients and could provide insight regarding the basis of lung injury and inflammation

The Systemic and Pulmonary Immune Response to Staphylococcal Enterotoxins

In response to environmental cues the human pathogen Staphylococcus aureus synthesizes and releases proteinaceous enterotoxins. These enterotoxins are natural etiologic entities of severe food poisoning, toxic shock syndrome, and acute diseases. Staphylococcal enterotoxins are currently listed as Category B Bioterrorism Agents by the Center for Disease Control and Prevention. They are associated with respiratory illnesses, and may contribute to exacerbation of pulmonary disease. This likely stems from the ability of Staphylococcal enterotoxins to elicit powerful episodes of T cell stimulation resulting in release of pro-inflammatory cytokines. Here, we discuss the role of the immune system and potential mechanisms of disease initiation and progression

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

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

Differential proteomic fingerprint of BAL fluid from SEA <i>vs</i>. vehicle alone injected mice.

<p>Mice were immunized as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040184#pone-0040184-g001" target="_blank">figure 1</a>. After 16 h BAL fluid was obtained and 2 mg of protein per sample was processed on the Beckman Coulter ProteomeLab PF 2D platform. Chromatofocusing was performed as a linear gradient from pH 8.0 to pH 4.0. Fractions were collected in 0.3 pH intervals, automatically reinjected for a second dimension on a C18 column at 50°C. (A) Two-dimensional proteomic maps of BAL fluid from SEA and vehicle alone immunized mice, representative of one out three experiments is shown. (B) Chromatograms of second dimension fractions from SEA treated mice were overlaid with their corresponding equivalents from vehicle alone-injected mice. Overlays revealed peaks present in two fractions of the SEA treated samples but undetectable in the vehicle alone injected samples. (C) The fractions were lyophilized and resolved by 4–15% SDS-PAGE. Bands detected by a protein-specific fluorescent dye were cut out, digested by trypsin, and identified by LC/MS/MS. (D) Peptides sequences were searched against the NCBInr database version 20060804 using the Proteometrics Software Suite and the Profound Search Algorithm. The data are representative of three independent biological replicates. Each sample run on the PF 2D was a pool of BAL fluid obtained from 5 mice.</p

Cytochrome b5 expression is restricted to bronchiole epithelium in lungs.

<p>Mice were immunized i.n. with 1 <i>µ</i>g of SEA or vehicle alone i.n., secondary (2°) challenge of either vehicle or SEA 48 h after the primary, and were sacrificed 5 h later (53 hrs total). (A) Immunostaining of the lungs was performed using a rabbit polyclonal antibody against cytochrome b5 (Cyt b5) and rabbit immunoglobulin control (Ig). The data are representative of three independent experiments with 2–4 mice per group, magnification 100X. (B) The percentage of positive cytochrome b5 staining per bronchiole is reported for each sample by analyzing 30 representative bronchioles from two separate experiments. The errors bars indicate the standard error of the mean. **p<0.001.</p

SEA induces classical pro-inflammatory factors in BALF.

<p>BALF obtained 2–88 h after i.n. SEA inhalation was tested for the presence of IL-6, TNF and SAA by ELISA. Plots show cytokine secretion, each symbol represents one mouse. BALF were collected from 10 experiments. The error bars indicate the standard error of the mean between the mice in one group. Statistical significance between the groups was evaluated by two-tailed Student’s <i>t</i> tests.</p

Overnight incubation with lactoferrin affects cell viability.

<p>Cell were treated with proteins at 5 mg/ml, except Exp. #4* (10 mg/ml), stimulated for 18 h as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6a and 6b</a>. Viability of cells was then assayed by flow cytometry using Mitoflow as a marker of mitochondrial integrity, the percentage of cell alive is reported in the table.</p><p>Overnight incubation with lactoferrin affects cell viability.</p