Efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS

The lymphatic vasculature is the natural pathway for the resolution of inflammation, yet the role of pulmonary lymphatic drainage function in sepsis-induced acute respiratory distress syndrome (ARDS) remains poorly characterized. In this study, indocyanine green–near infrared lymphatic living imaging was performed to examine pulmonary lymphatic drainage function in septic mouse models. We found that the pulmonary lymphatic drainage was impaired owing to the damaged lymphatic structure in sepsis-induced ARDS. Moreover, prior lymphatic defects by blocking vascular endothelial growth factor receptor-3 (VEGFR-3) worsened sepsis-induced lymphatic dysfunction and inflammation. Posttreatment with vascular endothelial growth factor-C (Cys156Ser) (VEGF-C156S), a ligand of VEGFR-3, ameliorated lymphatic drainage by rejuvenating lymphatics to reduce the pulmonary edema and promote draining of pulmonary macrophages and neutrophils to pretracheal lymph nodes. Meanwhile, VEGF-C156S posttreatment reversed sepsis-inhibited CC chemokine ligand 21 (CCL21), which colocalizes with pulmonary lymphatic vessels. Furthermore, the advantages of VEGF-C156S on the drainage of inflammatory cells and edema fluid were abolished by blocking VEGFR-3 or CCL21. These results suggest that efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS. Our findings offer a therapeutic approach to sepsis-induced ARDS by promoting lymphatic drainage function.


Western blotting
For western blots, lung tissues were lysed in chilled RIPA buffer containing protease inhibitor and phosphatase inhibitor for 30 minutes on ice. 10 μg protein was loaded for SDS-PAGE with standard western blotting procedures.The following primary antibodies and at a 1:1000 dilution: Anti-LYVE-1 antibodies (Abcam, Cat.No. ab183501), Anti-VEGFR3 antibodies (R&D System, Cat.No.
The proteins were detected using enhanced chemiluminescence (all GE Healthcare) and processed using Quantity One.

RT-qPCR
Total RNA was extracted with TRIzol Reagent (Invitrogen, Cat.No. A33250).cDNAs were synthesized with the PrimeScript RT Master Mix (Takara, RR036B) and subjected to RT-qPCR using specific primers.The sequences of the forward and reverse primers are shown in Supplementary Table 1.

Evans Blue exudation assay
Lymphatic barrier function was visualized in the Miles assay by exudation of Evans blue as previous reported (1).Briefly, mice were anesthetized and then 20 μL Evans Blue (1% in PBS) was injected into the footpad and left circulating for 30 minutes.
Subsequently, skin patches (10mm×5mm) close to the injection sites were incubated in formamide for 24 hours.Extracted Evans Blue and hemoglobin was measured spectrophotometrically at 610 and 450 nm, respectively.The Evans Blue/hemoglobin ratio is given.

Scanning electron microscope
For scanning electron microscope, ICG (green) was intratracheally poured into the lung by tracheal intubation, and then the lymphatic vessels which located near bronchus were stained into green own to the lymphatic drainage (Figure S1A).Subsequently, as previous described (2), these lymphatic vessels were isolated under the dissecting microscope and then cut into slices of 5 x 5 mm 2 , followed by fixation in 4% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2 hours at room temperature.Samples were incubated in 1% osmic acid for 1 hour, submerged in 2% tannic acid for 2 hours for electrical conduction, and progressively dehydration in increasing ethanol concentrations, followed by rinsing with isoamyl acetate.Then the specimens were dried in an Eiko HCP-2 Critical Point Dryer at 1,200 psi and 32°C for 1h, and sputter coated with gold by an Eiko IB-5 ionic sprayer meter.The specimens were examined at an accelerating voltage of 20kV using a Stereoscan 260 Hitachi SEM (Japan).

Supplemental Figure 1 :Supplemental Figure 2 :
At 24th hour after sepsis mice formation or the recombinant VEGF-C156S protein treatment, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg).Then, anesthetized mice were exsanguinated, and the lung, ear and pLNs tissue samples were immediately obtained.The samples were embedded in paraffin, stained with hematoxylin and eosin (H&E), and analyzed by using a light microscope (OLYMPUS,BX53F).Organ injury scores were determined by two independent investigators blinded to the research assignment and based on alveolar congestion, alveolar haemorrhage, alveolar wall/hyaline hyaline membrane thickness and inflammatory cell accumulation.In brief, 0 represented no injury, 1 represented slight injury (25%), 2 represented moderate injury (50%), 3 represented severe injury (75%), 4 represented very severe injury (almost 100%).The sum of the four variables represented the lung injury score (total score: 0-16)(3).For the ears, the swelling of skin scored by the thickness of ears, as inflammatory edema causes an increase in tissue.The lung tissue samples were homogenized with phosphate buffer to obtain the supernatant liquid for detecting the inflammatory factors.Briefly, the concentrations of MPO, IL-1β, IL-6, TNF-α in lung tissue homogenate and the serum were determined by using ELISA kits in accordance with the R&D system's protocols.Detecting the pulmonary lymphatic drainage by ICG imaging.(a) Indocyanine Green (ICG, 1mg/mL, 10μL, green), a near-infrared (NIR) fluorescent dye, was intratracheally poured into the lung by tracheal intubation.30 min later, isolating the lung.LV: lymphatic vessel, BV: blood vessel.(b) ICG drained into the pretracheal lymph nodes (pLNs) from the lung.The dermal lymphatic drainage function was impaired in LPS-induced sepsis.(a) Indocyanine Green (ICG) was injected intradermally into the footpad or the ear, and the movement of ICG from the injection site to the draining popliteal lymph nodes (dpLNs) or the deep cervical lymph nodes (dcLNs).(b) Procedure and timeline: At 6 hours after the intraperitoneal injection of LPS (10mg/kg), the sepsis model was induced.Meanwhile, ICG (0.1mg/mL, 6 μL) was intradermally injected into the skin.Fluorescence intensities were determined at 0 th hour, 24 th hour and 48 th hour using IVIS associated with the clearance rate to reflect the effect of tissue fluid clearance by lymphatic flow.The tissue samples were collected at 24 th hour.(c-f) ICG was intradermally injected into the footpad of different dose LPS-induced sepsis mice.The remaining ICG were determined at 0 th hour, 24 th hour and 48 th hour using IVIS, quantified, and presented as relative radiance (photons/sec per cm2/steradian).Panel c right bar: the color scale indicates fluorescence intensity.Upper yellow color indicates maximum fluorescence intensity, whereas lower red color indicates minimum fluorescence intensity.The ICG clearance rate at 24 th hour and 48 th hour (n=6 to 7; representative data from three independent experiments).(g-h) At 24 th hour, ICG (0.1mg/mL, 6 μL) was intradermally injected into the footpad.Fluorescence intensities were determined in dpLNs at 30 minutes after the injection, quantified, and presented as relative radiance (Control n=7, LPS n=7; representative data from three independent experiments).(i-l) ICG (0.1mg/mL, 6 μL) was intradermally injected into the skin of ear and quantified by IVIS.The ICG clearance rate at 24 th hour and 48 th hour (Control n=6, LPS n=5; representative data from three independent experiments).(m-n) At 24 th hour, ICG was intradermally injected into the skin of ear.Fluorescence intensities were determined in dcLNs at 30 minutes after the injection (Control n=5, LPS n=5; representative data from three independent experiments).p values were calculated by a one-way ANOVA with Tukey's multiple comparison test or two-tailed paired or unpaired Student's t-test.Sequences (5' to 3')