Methods
Animals and treatment
For all experiments, mice between 6 to 8 weeks of age and 17 g to 20 g were used including males and females in equal proportions. The following strains were utilized: Inducible Cre (UBC-CreERT2) [14] (The Jackson Laboratory Stock #007001), RSPO2 flox (a gift from Dr. Kurt Hankenson, University of Michigan), and C57BL/6 mice. No statistical method was used to predetermine sample size in any of the animal studies. The experiments were not randomized, and the investigators were not blinded to allocation during the experiments and outcome assessments.
Cre recombination in vivo
Mice were injected intraperitoneally with tamoxifen (TM) in 100 µl of Mazola® corn oil (1 mg/g body wt) once per day, every other day for a total of 3 doses. For all analyses, tissues were collected 2 days after the last TM injection.
Animal euthanasia
Mice were placed into a closed chamber and exposed to isoflurane (Midwest Veterinary Supply) applied to compacted cotton balls until roughly 1 min after breathing stopped, followed by cervical dislocation, as approved by IACUC.
Fibroblast isolation
Fibroblasts were isolated according to our general lung cell isolation protocol previously described [15] with slight modifications: 1 × 107 disaggregated lung cells were plated into a Corning® 6-well clear polystyrene flat-bottom microplate (Millipore Sigma) in DMEM + 20% CC + P/S and grown in a 37 °C incubator for 9 days without passaging with media changes on days 3 and 6 before harvesting cells for mRNA or ICC analysis. For Cre-induced recombination in cultured fibroblasts, cells were treated with (4 µM) of 4-Hydroxytamoxifen (4-OHT) dissolved in dimethyl sulfoxide (DMSO; Santa Cruz Biotechnology), once per day, every other day for 5 days. For qPCR and ICC analyses, fibroblast samples were collected 2 days after the last 4-OHT treatment.
BALF collection
BALF was collected according to the protocol previously described [16], followed by cytospin preparation.
Cytospins
For both cultured fibroblasts and BALF, cells were centrifuged at 570×g for 5 min, followed by aspiration of supernatant, and cell pellets were resuspended in 1 ml of PBS solution and fixed onto slides (Fisherbrand™) at 570 rpm for 4 min on a Cytospin 2 (Shandon).
Antibody staining
Immunostaining was performed as previously described [16]. The following primary antibodies were used: goat anti-myeloperoxidase (MPO) (1:200 dilution; R&D Systems), rabbit anti-RSPO2 (1:200 dilution; Proteintech). The following secondary antibodies were used: Alexa Fluor 488-conjugated donkey anti-goat (1:1000, Thermo Fisher Scientific), Alexa Fluor 568-conjugated donkey anti-rabbit (1:1000, Thermo Fisher Scientific).
Quantification of immunostaining
Mosaic images of cytospins were generated from multiple 20 X fields on an upright fluorescence microscope (Leica DMi8) and tiled in LAS X software. The number of cells staining positive for the relevant antibody was manually counted and calculated as a fraction of total DAPI + cells. We quantified at least three fields per slide, each containing ≥ 300 individual cells.
Quantitative PCR (qPCR) analysis
RNA was isolated using the RNeasy™ (Qiagen) kit. mRNA was revered transcribed into cDNA using iScript™ Reverse Transcription Supermix (BioRad). Total RNA input for cDNA synthesis was standardized within each experiment to the RNA isolate with the lowest concentration as measured by Nanodrop (Thermo Fisher Scientific). RT-PCR reactions were performed using SsoAdvanced™ Universal SYBR® Green Supermix (Biorad) and run on an Applied Biosystems QuantStudio 6 Real-Time PCR System (Thermo Fisher Scientific).
FITC-dextran permeability assay
The permeability assay was performed as described in the literature [17, 18]. Mice were anaesthetized with isoflurane and administered 40 µl FITC-dextran (10 mg/kg body weight) intranasally. After a 30-min wait to allow FITC-dextran to circulate in the blood, blood was collected via cardiac puncture, and fluorescence intensity was determined using a spectrophotometer (BioTek).
Statistical analysis
All statistical calculations were performed using GraphPad Prism. Mann–Whitney test was used to determine significance. A P value of less than 0.05 was considered significant.
PCR and qPCR primers
Genotyping primers
Rspo2-floxA-Forward: ACTCTTACTGCCTGGGATCCTCATT
Rspo2-floxB-Reverse: CTTCTTCTGAGCACCATCTGC
qPCR primers
GAPDH Forward: AGGTCGGTGTGAACGGATTTG
GAPDH Reverse: TGTAGACCATGTAGTTGAGGTCA
RPL37 Forward: CTCGGAGGTTACGGGACTC
RPL37 Reverse: CTTGCCCTCGTAGGTAATGGG
RPL19 Forward: ATG TAT CAC AGC CTG TAC CTG
RPL19 Reverse: TTC TTG GTC TCT TCC TCC TTG
MPO Forward: AGTTGTGCTGAGCTGTATGGA
MPO Reverse: CGGCTGCTTGAAGTAAAACAGG
RSPO2 Forward: AGACGCAATAAGCGAGGTGG
RSPO2 Reverse: CTGCATCGTGCACATCTGTT
Results
Infiltration of neutrophils into bronchoalveolar lavage fluid following RSPO2 deletion
Given that tissue repair often recapitulates features of embryonic development, where RSPO2 is critical, we generated UBC-CreERT2/RSPO2flox/flox mice to pursue the hypothesis that RSPO2 deletion would impact lung regeneration. We first confirmed successful recombination of the RSPO2 allele in adult mice after TM treatment (Fig. 1a). Additionally, we isolated lung fibroblasts from these animals, treated with 4-OHT in vitro to induce recombination, and confirmed reduction in RSPO2 transcript via qPCR and immunostaining (Fig. 1b–d). Before the originally planned injury experiments were initiated, we examined the lavage fluid of RSPO2 deleted mice to ensure normal levels of immune cells as determined by cytospin cell analysis. Unexpectedly, we observed MPO-expressing cells, a definitive neutrophil marker [19], in the BALF of RSPO2−/− mice at a significantly higher percentage compared to RSPO2+/+ mice (Fig. 2a, b). qPCR analysis demonstrated significantly higher MPO expression in BALF cells in RSPO2−/− mice compared to RSPO2+/+ mice (Fig. 2c), confirming the increase of infiltrated neutrophils. This indicates RSPO2−/− mice exhibit elevated neutrophil egress into the alveolar space compared to RSPO2+/+ mice in terms of both increased MPO-expressing cells and higher MPO mRNA expression in BALF cells.
RSPO2 deletion increases lung barrier permeability
Because neutrophils must exit circulation through the vasculature before translocation into the alveolar lumen [13], we hypothesized that RSPO2 deletion might induce endothelial disruption to facilitate the observed egress of neutrophils into the bronchoalveolar space. To assess lung permeability resulting from endothelial disruption, we administered FITC-dextran via intranasal instillation [16,17,18] and measured fluorescence in blood plasma after 30 min. A significant increase in plasma dextran concentration was observed in RSPO2−/− mice compared to identically treated RSPO2+/+ mice (Fig. 3a, b). Taken together, these data indicate that RSPO2 deletion increases lung barrier permeability.
Discussion
While RSPO2 expression in the embryonic lung mesenchyme is essential for normal lung development [2], whether RSPO2 expression in the adult lung is relevant in tissue homeostasis or repair has not been investigated. Our studies indicate an unexpected and biologically important role for RSPO2 in the lung as a regulator of neutrophil homeostasis and endothelial barrier function. Deletion of RSPO2 induces vascular leak and neutrophil accumulation in the airspace, indicating a novel role for R-Spondin signaling in these contexts.
To the best of our knowledge, RSPO2 has not been previously implicated in neutrophil homeostasis/chemotaxis. Given the well described role of R-Spondins in potentiating Wnt signaling, we presume dysregulation of Wnt is the likely driver behind this phenotype. For example, Wnt5a is known to activate noncanonical Wnt pathways and activate neutrophil chemotaxis [20], and although involvement of R-spondins has not been investigated, our work supports their possible involvement.
RSPO2’s role in regulation of vascular permeability is not entirely without precedent. In the adult gastrointestinal tract, another RSPO family member, RSPO3, tightens endothelial cell junctions, limiting fluid egress from the circulation [21]. Given high expression of RSPO2 in the developing lung, RSPO2 may also play a role in the dynamic regulation of microvascular permeability that occurs at birth during the transition to air breathing [22, 23]. It is worth considering whether RSPO2, by increasing barrier integrity, may act to counterbalance other factors which decrease integrity/induce permeability, akin to how endothelin and nitric oxide act as natural counterparts to regulate vasoconstriction and vasodilation, respectively [24].
While these studies highlight potentially important new roles for RSPO2, there are many outstanding questions which require further study. First, we presume that the combination of barrier dysfunction and a second mechanism, likely involving neutrophil chemokine dysregulation, explains the appearance of neutrophils in BALF as opposed to nonspecific accumulation of circulating immune cells. Further studies are also needed to understand whether neutrophils are being actively recruited or whether they arrive in the alveolar space passively. Moreover, because we employed a broadly expressed Cre driver, whether the phenotypes described here are cell autonomous or non-autonomous is unknown. Based on developmental studies [2, 25] we presume the lung mesenchyme is the predominant source of RSPO2 which acts primarily in a paracrine fashion (i.e. from mesenchymal cells to endothelial cells and hematopoietic cells), but this should be formally investigated. It is also possible that autocrine RSPO2 deletion in the endothelium itself leads to the vascular phenotype. Likewise, RSPO2 deletion in the neutrophils themselves could cause spurious activation. Our findings here indicate careful, cell type-specific studies should be performed to elucidate the range of RSPO2 functions in the adult lung and beyond.
Further investigations at the molecular level will be necessary to shed more light on the exact molecular mechanisms by which RSPO2 may regulate neutrophil migration, chemoattractant responsiveness, and basal lung barrier function. Ultimately, these initial findings should seed larger efforts to elucidate specific roles for RSPO2 in lung homeostasis and disease.