Lipopolysaccharides induce a RAGE-mediated sensitization of sensory neurons and fluid hypersecretion in the upper airways

Thoracic dorsal root ganglia (tDRG) contribute to fluid secretion in the upper airways. Inflammation potentiates DRG responses, but the mechanisms remain under investigation. The receptor for advanced glycation end-products (RAGE) underlies potentiation of DRG responses in pain pathologies; however, its role in other sensory modalities is less understood. We hypothesize that RAGE contributes to electrophysiological and biochemical changes in tDRGs during inflammation. We used tDRGs and tracheas from wild types (WT), RAGE knock-out (RAGE-KO), and with the RAGE antagonist FPS-ZM1, and exposed them to lipopolysaccharides (LPS). We studied: capsaicin (CAP)-evoked currents and action potentials (AP), tracheal submucosal gland secretion, RAGE expression and downstream pathways. In WT neurons, LPS increased CAP-evoked currents and AP generation, and it caused submucosal gland hypersecretion in tracheas from WT mice exposed to LPS. In contrast, LPS had no effect on tDRG excitability or gland secretion in RAGE-KO mice or mice treated with FPS-ZM1. LPS upregulated full-length RAGE (encoded by Tv1-RAGE) and downregulated a soluble (sRAGE) splice variant (encoded by MmusRAGEv4) in tDRG neurons. These data suggest that sensitization of tDRG neurons contributes to hypersecretion in the upper airways during inflammation. And at least two RAGE variants may be involved in these effects of LPS.

2 Lawn, NJ, USA) buffer (10% TFE, 100 mM ammonium bicarbonate (ABC)). Proteins were digested in-solution using an in-house developed protocol. Briefly, 45 µl of each protein sample was placed in a 1.5 ml tube and diluted with 5 µl of 1 M ABC buffer (Fisher Scientific, Fair Lawn, NJ, USA) and 50 µl TFE to denature proteins. The samples were treated with 1 µl of 1M DTT (dithiothreitol) (MP Biomedicals, Solon, OH, USA) while shaking at 300 RPM (Eppendorf Thermomixer, Eppendorf, Mississauga, ON, Canada) at 60 o C for 60 min to reduce disulfide bonds.
Next, samples were alkylated with 100 µl of 110 mM iodoacetamide (IAA; Fisher Scientific, Fair Lawn, NJ, USA) at 37 o C for 30 min on a shaker, covered with aluminum foil, to prevent further disulfide bond formation. The samples were dried in a speedvac (Labconco, Kansas City, MO, USA). Proteins in the samples were treated with 1 ml cold acetone followed by refrigeration at -80 o C for 60 min to eliminate salts and other interfering compounds (e.g. detergents), which can prevent digestion. The samples were centrifuged twice at 18000 x g for 30 min and acetone was carefully removed. Next, samples were dried in a speedvac and a buffer-containing trypsin (Promega Corporation, Madison, WI, USA) solution (50 ng/µl trypsin in 1 mM HCl (hydrochloric acid) /100 mM ABC) was added to the samples in a 40:1 protein:trypsin ratio. The samples were incubated in a shaker at 300 RPM overnight at 37 o C. Trypsin buffer at the same ratio was added again in the morning to ensure complete digestion of proteins into peptides. After 2 hrs of further incubation at 37 o C, digested peptides were dried in speedvac and stored at -80 o C until further analysis.
The digested protein samples were dissolved in 200 µl of SCX reconstitution solution (pH 3). The 3 samples were then loaded on to SCX SpinTip and centrifuged at 2000 x g for 6 min. To enhance peptide binding to SCX SpinTip samples were reloaded and centrifuged 3 more times. The flow through at the end of the step was transferred to a 1.5 ml tube for MS analysis. Peptides bound to SCX column were eluted using stepwise concentrations (in M): 20, 40, 60, 80, 100, 150, 250, 500 of ammonium formate (Sigma-Aldrich) in 10% acetonitrile (ACN; Fisher Scientific) at pH 3. 150 µl of ammonium formate (in increasing concentrations) was added to the SpinTip and centrifuged at 2000 x g for 6 min. The flow through was collected after every centrifugation and transferred to 1.5 ml tube. All flow throughs were dried in speedvac and stored at -80 o C.

Protein Identification.
Tandem mass spectra were extracted from raw data and were processed against the NCBI (National Center for Biotechnology Information) non-redundant Mus musculus database as well as custom database (containing all known mouse RAGE protein isoforms), using Spectrum Mill (Agilent Technologies) as the database search engine. Search parameters included a fragment mass error of 50 parts per million (ppm), a parent mass error of 20 ppm, trypsin cleavage specificity, and carbamidomethyl as a fixed modification of cysteine. In addition, four stages of database search in variable modification mode were carried out with different sets of variable modifications. In the first stage, carbamylated lysine, oxidized methionine, pyroglutamic acid, deamidated asparagine and phosphorylated serine, threonine, and tyrosine were set as variable modifications. In the second stage, validated hits from the first stage were searched using the following variable modifications: acetyl lysine, oxidized methionine, pyroglutamic acid, deamidated asparagine, and phosphorylated serine, threonine, and tyrosine.

5
The validated hits from the second stage were searched using semi-trypsin non-specific Cterminus, yielding the third stage validated hits, which were subsequently searched using semitrypsin non-specific N-terminus (fourth stage), with no other variable modifications specified.
After each stage, Spectrum Mill validation was performed at peptide and protein levels (1% false discovery rate, FDR), and spectral counts and intensities were used to report relative quantitation of proteins. The Mass Profiler Professional (MPP, version 15.0, Agilent, Santa Clara, CA, USA) software was used for statistical analysis using one-way ANOVA. A cut-off value of p < 0.05 and the Benjamini and Hochberg FDR set at < 1% were used to obtain statistically significant results.
In addition, a fold change (FC) of ≥ 2 and < 0.5 in spectral intensities with respect to control were considered to classify proteins as up-and down-regulated, respectively, in LPS samples.