Inflammatory Cytokine Elaboration Following Secondhand Smoke (SHS) Exposure Is Mediated in Part by RAGE Signaling.

The receptor for advanced glycation end products (RAGE) is a key contributor to immune and inflammatory responses in myriad diseases. RAGE is a transmembrane pattern recognition receptor with a special interest in pulmonary anomalies due to its naturally abundant pulmonary expression. Our previous studies demonstrated an inflammatory role for RAGE following acute 30-day exposure to secondhand smoke (SHS), wherein immune cell diapedesis and cytokine/chemokine secretion were accentuated in part via RAGE signaling. However, the chronic inflammatory mechanisms associated with RAGE have yet to be fully elucidated. In this study, we address the impact of long-term SHS exposure on RAGE signaling. RAGE knockout (RKO) and wild-type (WT) mice were exposed to SHS using a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months. SHS-exposed animals were compared to mice exposed to room air (RA) only. Immunoblotting was used to assess the phospho-AKT and phospho-ERK activation data, and colorimetric high-throughput assays were used to measure NF-kB. Ras activation was measured via ELISAs. Bronchoalveolar lavage fluid (BALF) cellularity was quantified, and a mouse cytokine antibody array was used to screen the secreted cytokines. The phospho-AKT level was decreased, while those of phospho-ERK, NF-kB, and Ras were elevated in both groups of SHS-exposed mice, with the RKO + SHS-exposed mice demonstrating significantly decreased levels of each intermediate compared to those of the WT + SHS-exposed mice. The BALF contained increased levels of diverse pro-inflammatory cytokines in the SHS-exposed WT mice, and diminished secretion was detected in the SHS-exposed RKO mice. These results validate the role for RAGE in the mediation of chronic pulmonary inflammatory responses and suggest ERK signaling as a likely pathway that perpetuates RAGE-dependent inflammation. Additional characterization of RAGE-mediated pulmonary responses to prolonged exposure will provide a valuable insight into the cellular mechanisms of lung diseases such as chronic obstructive pulmonary disease.

Decreased Expression of Pulmonary Homeobox NKX2.1 and Surfactant Protein C in Developing Lungs That Over-Express Receptors for Advanced Glycation End-Products (RAGE).

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobin superfamily prominently expressed by lung epithelium. Previous experiments demonstrated that over-expression of RAGE by murine alveolar epithelium throughout embryonic development causes neonatal lethality coincident with significant lung hypoplasia. In the current study, we evaluated the expression of NKX2.1 (also referred to as TTF-1), a homeodomain-containing transcription factor critical for branching morphogenesis, in mice that differentially expressed RAGE. We also contextualized NKX2.1 expression with the abundance of FoxA2, a winged double helix DNA binding protein that influences respiratory epithelial cell differentiation and surfactant protein expression. Conditional RAGE over-expression was induced in mouse lung throughout gestation (embryonic day E0-18.5), as well as during the critical saccular period of development (E15.5-18.5), and analyses were conducted at E18.5. Histology revealed markedly less lung parenchyma beginning in the canalicular stage of lung development and continuing throughout the saccular period. We discovered consistently decreased expression of both NKX2.1 and FoxA2 in lungs from transgenic (TG) mice compared to littermate controls. We also observed diminished surfactant protein C in TG mice, suggesting possible hindered differentiation and/or proliferation of alveolar epithelial cells under the genetic control of these two critical transcription factors. These results demonstrate that RAGE must be specifically regulated during lung formation. Perturbation of epithelial cell differentiation culminating in respiratory distress and perinatal lethality may coincide with elevated RAGE expression in the lung parenchyma.