Objective R-spondin 2 (RSPO2) is required for lung morphogenesis, activates Wnt signaling, and is upregulated in idiopathic lung fibrosis. Our objective was to investigate whether RSPO2 is similarly important in homeostasis of the adult lung. While investigating the characteristics of bronchoalveolar lavage in RSPO2-deficient (RSPO2-/-) mice, we observed unexpected changes in neutrophil homeostasis and vascular permeability when compared to control (RSPO2+/+) mice at baseline. Here we quantify these observations to explore how tonic RSPO2 expression impacts lung homeostasis. Results Quantitative PCR (qPCR) analysis demonstrated significantly elevated myeloperoxidase (MPO) expression in bronchoalveolar lavage fluid (BALF) cells from RSPO2-/- mice. Likewise, immunocytochemical (ICC) analysis demonstrated significantly more MPO+ cells in BALF from RSPO2-/- mice compared to controls, confirming the increase of infiltrated neutrophils. We then assessed lung permeability/barrier disruption via Fluorescein Isothiocyanate (FITC)-dextran instillation and found a significantly higher dextran concentration in the plasma of RSPO2-/- mice compared to identically treated RSPO2+/+ mice. These data demonstrate that RSPO2 may be crucial for blood-gas barrier integrity and can limit neutrophil migration from circulation into alveolar spaces associated with increased lung permeability and/or barrier disruption. This study indicates that additional research is needed to evaluate RSPO2 in scenarios characterized by pulmonary edema or neutrophilia.

Objective: R-spondin 2 (RSPO2) is required for proper lung morphogenesis. Our objective was to investigate whether RSPO2 is similarly important in homeostasis of the adult lung. Unexpectedly, we observed changes in neutrophil migration and lung vascular permeability in RSPO2-deficient (RSPO2 -/-) mice compared to RSPO2 control (RSPO2 +/+ ) mice, independent of experimental injury/challenge.Here we use multiple methods to quantify these observations to further understand how tonic RSPO2 expression regulates lung homeostasis.Results: Quantitative PCR (qPCR) analysis demonstrated significantly higher myeloperoxidase (MPO) expression in bronchoalveolar lavage fluid (BALF) cell content from RSPO2 -/mice compared to RSPO2 +/+ mice. Immunocytochemical (ICC) analysis likewise demonstrated significantly more MPO+ cells in BALF from RSPO2 -/mice compared to RSPO2 +/+ mice, confirming the increase of infiltrated neutrophils. We then assessed lung permeability/barrier disruption via Fluorescein isothiocyanate (FITC)-dextran instillation and found a significantly higher dextran concentration in the plasma of RSPO2 -/mice compared to identically treated RSPO2 +/+ mice. These data demonstrate that RSPO2 may be crucial for lung barrier integrity and can facilitate an increase in neutrophil migration from circulation into alveolar spaces due to increased lung permeability/barrier disruption. Our studies suggest additional research is needed to evaluate RSPO2 in scenarios exhibiting either pulmonary edema or neutrophilia.