Influenza Induces Lung Lymphangiogenesis Independent of YAP/TAZ Activity in Lymphatic Endothelial Cells

The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. The mechanisms for organ-specific LEC responses to environmental cues are not well understood. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases 2-fold at 7 days post-influenza infection (dpi) and 3-fold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. Lastly, using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.


Introduction
The pulmonary lymphatic vasculature plays a number of crucial roles in orchestrating the response to infection and tissue injury (1).These vessels are lined with specialized lymphatic endothelial cells (LECs), which maintain their identity through the constitutive expression of Prospero homeobox protein 1 (PROX1) (2) and are characterized by vascular endothelial growth factor receptor 3 (VEGFR3) expression (3)(4)(5).Lymphatics modulate interstitial uid drainage and transport immune cells and antigen to lymph nodes (6).Signaling between LECs and immune cells also directs chemotaxis (7) and immune cell proliferation (8).During respiratory tract in ammation, these functions are essential, as excessive tissue uid buildup or an ineffective immune response could severely compromise lung function.
New lymphatic vessel growth, or lymphangiogenesis, can be observed in in amed tissue (9).Although this has been shown in the respiratory system in response to several types of injury, including bacterial infection (10,11) and pulmonary brosis (12,13), it is unknown whether it occurs in response to acute viral infections such as in uenza.
In uenza viruses target the respiratory epithelium, triggering severe in ammation (14).In spite of advances in prophylaxis and treatment approaches, in uenza infection is associated with 14% of acute respiratory disease-related hospitalizations (15) and an estimated 290,000-650,000 annual respiratory deaths worldwide (16).Concentrating on the host's response, which is pivotal in determining in uenza's severity and outcome, opens avenues for novel therapeutic interventions.In this context, lymphatic vessel dysfunction might weaken tissue resilience and impede pathogen removal.Facilitating pulmonary lymphatic responses could theoretically, therefore, enhance viral elimination, counteract damaging immune dysregulation and strengthen uid removal in severe cases.The Hippo pathway, known for its high conservation and central role in cell proliferation, organ development and morphogenesis, and tissue responses to injury could be key in guiding lymphatic endothelial cell behavior during in uenza infection (17)(18)(19).
Mechanical stimuli, such as stretch and shear forces are common environmental cues that modulate Hippo signaling (17,18,20,21).In Hippo signaling, two downstream effectors jointly regulate gene expression: Yes-associated protein (YAP), encoded by the Yap1 gene, and Transcriptional co-activator with PDZ-binding motif (TAZ), encoded by the Wwtr1 gene.YAP and TAZ protein activity depend on intracellular localization.Nuclear YAP and TAZ regulate a wide variety of target genes, whereas cytoplasmic YAP and TAZ are targeted for degradation (22).Notably, YAP/TAZ depletion (23,24) or hyperactivation (23) in LECs during embryonic development results in structurally aberrant and poorly functional lymphatics and lethality, highlighting its importance in early development of the lymphatic system.In adults, however, intact Hippo signaling in LECs is less critical for maintaining lymphatic integrity during homeostasis (23).The functional role(s) of Hippo signaling in adult lung LECs, particularly during in ammatory responses, remain unexplored.
In blood endothelial cells, mechanosensitive signaling (20,21) and molecular signals such as vascular endothelial growth factor A (VEGF-A) (25)(26)(27) act through the Hippo pathway to regulate cell proliferation.The major pro-lymphangiogenic growth factor, VEGF-C, also in uences Hippo signaling (23,24).While increased uid in ux and altered levels of cytokines and growth factors are key characteristics of the lung's in ammatory response, to our knowledge, no study has investigated what role YAP/TAZ signaling might play in pulmonary lymphatic vessels as they respond to in uenza-induced in ammation.
Here we show that the pulmonary lymphatic system undergoes extensive dilation and lymphangiogenesis in a mouse model of in uenza infection.We show by lineage tracing that the expanded lymphatic network in the lung derives from existing LECs.We also demonstrate that there is a signi cantly higher number of proliferating pulmonary LECs during in uenza.The resulting lymphatic network exhibits increased uid drainage from the lung to mediastinal lymph nodes.In view of these ndings, we investigated the role of the Hippo signaling pathway in LECs during in uenza.Our results demonstrate that LEC-speci c deletion of these Hippo pathway effectors revealed that YAP/TAZ signaling is dispensable for pulmonary lymphangiogenesis, lymphatic drainage and host response in the adult lung during in uenza.These ndings were unanticipated, as they suggest that Hippo-dependent signaling is not crucial for these processes in adults.This discovery yields new avenues for understanding the mechanisms underlying pulmonary responses to in uenza A infection and the regulation of lymphatic function in adult lungs.

Results
Pulmonary lymphangiogenesis in in uenza pneumonia.
To investigate the pulmonary lymphatic vessel responses to in uenza infection, we intratracheally infected the left lobe of mice with PR8 in uenza and harvested left lungs at 3-, 7-and 21-days postinfection (dpi) for para n-embedding and sectioning.Mice were weighed daily during the time-course of infection to assess morbidity (Supp.Figure 1).We then stained for VEGFR3 in lung sections of in uenzainfected and control mice to identify lymphatic vessels.We observed a signi cant and sustained enlargement in the diameter of VEGFR3-positive vessels in the lung during in uenza infection as soon as 3 dpi and until at least 21 dpi (Fig. 1a and b).This indicated that the lymphatic vessels are dilated in response to in uenza infection.
To determine whether lymphangiogenesis accompanied vessel dilation, we quanti ed the lung LEC population by labeling LEC nuclei during a time-course of in uenza infection.In order to do this, we stained for PROX1 at 3, 7 and 21 dpi and enumerated LECs in each histological section comparing in uenza-infected lungs with controls.This experiment revealed a doubling of LECs by 7 dpi that continued to increase through 21 dpi (Fig. 2a and b).Our ndings indicate that in uenza infection not only leads to the enlargement of existing lymphatic vessels but also stimulates the expansion of LEC number.
Pulmonary lymphangiogenesis in in uenza is driven by LEC proliferation.
To identify the source of new LECs in the lymphatic network in in uenza-infected lungs, we employed two independent approaches.Firstly, to label nascent DNA incorporation, 5-Ethynyl-2-deoxyuridine (EdU) was administered to mice during in uenza infection.Lungs were harvested at 7 dpi and immuno uorescently co-stained for PROX1 and EdU (Fig. 3a and b).While proliferating LECs were very rarely observed in control tissues, approximately 20% of LECs in in uenza-infected lungs had incorporated EdU by 7 dpi (3b).Secondly, we investigated the possibility that an exogenous progenitor cell might play a role in in uenza-induced lymphangiogenesis.To address this question, tamoxifen-induced LEC lineage labeling was performed in PROX1-CreER T2 /tdTomato mice prior to in uenza infection.The proportion of tdTomato positive LECs observed during in uenza infection was then compared to control lungs.Despite the increased number of LECs observed during in uenza (data not shown), there was a similar proportion of lineage labeled cells at 7 dpi (Fig. 3c and d).These ndings demonstrate that PROX1-negative progenitor cells are not contributing to lymphangiogenesis during in uenza.However, we cannot exclude the possibility of a PROX1-positive progenitor.Notably, EdU uptake in LECs was not observed in the liver, heart, or esophageal tissues, indicating that LEC proliferation during in uenza was speci c to the lungs (data not shown).Taken together, these results suggest endogenous lung LEC proliferation in response to in uenza infection contributes to LEC expansion.Lymphatic transport in in uenza pneumonia.
Consistent with published literature, in uenza infection leads to pronounced in ammation, and pulmonary edema.In this regard, we observed signi cantly higher lung wet-to-dry weight ratios during in uenza infection as compared to controls, indicative of pulmonary edema (Fig. 4a).To investigate the functional properties of the expanded lymphatic network, we instilled a 10 kDa uorescent dextran molecule into the left lung airspaces and measured uorescence in the lung-draining mediastinal lymph node (mLN) (Fig. 4b and c).We observed a signi cantly higher uorescent signal in the mLNs of in uenza-infected mice as compared to control at 15 minutes.There was minimal uorescent signal detected in non-draining inguinal LNs indicating local lymphatic drainage as opposed to blood vessel drainage was primarily responsible for transport of dextran (data not shown).Collectively, these ndings indicate that the expanded lymphatic network in the in uenza-infected lung is associated with enhanced lymphatic transport.
Role of Hippo signaling during in uenza-induced pulmonary lymphangiogenesis.
The Hippo pathway is a fundamental mediator of cell proliferation during development and is activated in response to injury and mechanical cues.Notably, Hippo signaling in LECs is critical for lymphangiogenesis and lymphatic patterning during embryonic development (23,24).To determine the role for Hippo signaling in LECs during in uenza-induced lymphangiogenesis, we utilized a previously published model of Hippo pathway deletion in LECs (Prox1-CreERT2/Yap1(YAP)-/Wwtr1(TAZ)-, hereafter YAP/TAZ △LEC ).In all experiments, Cre(-) littermates given tamoxifen were used as controls.
First, we validated YAP and TAZ depletion in LECs using immuno uorescent staining of lung histologic sections and by validating the presence of the oxed YAP and TAZ alleles after tamoxifen administration (Supp.Figures 2 and 3).To con rm e cient PROX1-Cre-mediated recombination, we analyzed tdTomato expression in LECs in mice at least 2 weeks after tamoxifen administration to Prox1-CreER T2 / TdTomato mice (Supp.Figure 4).Next, we analyzed in uenza-infected lungs for differences in histologic lymphatic phenotype, including lymphatic vessel diameter measurement and LEC enumeration as previously described.We identi ed no signi cant differences in these two parameters between Cre(+) and Cre(-) YAP/TAZ △LEC littermates, either at baseline, 7 or 16 dpi (Fig. 5a-f).Similarly, no differences in Prox1 or Flt4 mRNA were observed in whole lung homogenates obtained from either Cre(+) or Cre(-) YAP/TAZ △LEC littermates at 7 dpi (Fig. 5g).
To determine whether there were differences in pulmonary edema in the context of Hippo deletions, we measured lung wet-to-dry weight ratios during in uenza as well as control conditions, and found no difference between Cre(+) and Cre(-) YAP/TAZ △LEC littermates (Fig. 5h).
The dextran transport assay was utilized to interrogate the functionality of lymphatics for passive drainage in uninfected lungs.In these experiments, no signi cant difference was observed in uorescent signal measured in the lung-draining mLNs of Cre(+) vs Cre(-) YAP/TAZ △LEC littermates at baseline or at 7dpi (Fig. 5i and j).Collectively, the targeted deletion of YAP and TAZ in LECs did not affect lymphangiogenesis or lung lymphatic drainage at baseline or during in uenza pneumonia.
Infection severity and in ammatory response after deletion of YAP and TAZ in LECs.
Infection severity (28) and in ammatory responses (9,29) are known to be affected by aberrant lymphatic function.To address this issue, we rst compared survival and weight loss curves after in uenza pneumonia in Cre(+) and Cre(-) YAP/TAZ △LEC littermates.No mice reached the humane endpoint in either group, and we found no differences in weight change during or after in uenza infection between the two Cre genotypes (Fig. 6a).We then assessed infection severity by plaque assay and qRT-PCR for in uenza nucleoprotein (NP) mRNA in murine lungs at 7 dpi and found no difference in these readouts between Cre genotypes (Fig. 6b and c).We also characterized the in ammatory response to in uenza in the lung at 7 and 16 dpi in both Cre genotypes.These timepoints mark two critical phases: the lowest point of weight loss and the subsequent return to baseline weight after in uenza infection.Detailed in ammation scoring and histopathologic characterization was provided by a veterinary pathologist who was blinded to genotypes.In summary, there were no apparent differences due to YAP/TAZ-deletion in morphological histopathology as assessed by the percent area of consolidation or semiquantitative pathology score (Fig. 6d and e, Supp.Tables 1 and 2).Lastly, to evaluate differences in immune cell transit between the lung and the lung-draining mLN, we designed an 11-color ow cytometry panel to determine the immunophenotype of the lymph node at 2 and 7 dpi.Using markers for T-cells (CD4, CD8) and B-cells (CD19) as well as markers for dendritic cell subtypes (Ly6C, CD11c, CD11b and CD103), we were able to de ne cell populations known to be important in the developing adaptive immune response, for which a functional lymphatic vasculature is known to be crucial (28, 30).While we did observe predictable differences in the mLN immunophenotype between 2 and 7 dpi, we identi ed no differences in any of the de ned immune cell populations between Cre(+) or Cre(-) YAP/TAZ △LEC littermates (Fig. 7a and b).

Discussion
Our studies demonstrate that the pulmonary lymphatic system expands signi cantly during severe in uenza, via both dilation and lymphangiogenesis.This is a stark change from the quiescent state of the lymphatic plexus during homeostasis (31) and is reminiscent of the rapid lymphatic growth seen in development (32).Furthermore, our ndings demonstrate that lymphangiogenesis occurs primarily through proliferation of existing LECs, without an apparent involvement of an exogenous precursor.Moreover, we found that following this expansion, the lymphatic vasculature displays enhanced uid transport.
The in ammation resulting from in uenza infection may trigger responses in LECs through mechanical signals.For example, alterations in extracellular matrix (ECM) composition may modulate matrix stiffness, as has been reported in pulmonary brosis (33).Higher uid volume within the lymphatics can increase circumferential stretch (34).As YAP/TAZ intracellular localization is sensitive to such physical changes (35)(36)(37), we investigated whether the Hippo pathway plays a part in orchestrating the lymphatic response to in uenza-induced in ammation.
We found that during in uenza-induced lymphangiogenesis, the targeted deletion of YAP and TAZ in PROX1-expressing cells had no effect on uid transport from the lung to the draining lymph nodes at baseline or during in uenza infection.Lymphatic vessel diameter, LEC number, Prox1 and Flt4 mRNA transcription and lung wet-to-dry weight ratios at 7 dpi were also unaffected by LEC-speci c YAP/TAZ deletion.
Previous work has yielded con icting ndings regarding the regulatory relationship between YAP/TAZ and PROX1.Using PROX1-driven, Cre-mediated deletion of YAP and TAZ, Cho et.al. nd that YAP and TAZ activity opposes PROX1 expression during development, limiting lymphangiogenesis (23).Conversely, in vitro experiments show that both YAP and TAZ knockdown and hyperactivation are associated with diminished PROX1 expression (24).In our studies, the lymphatic remodeling we observed in response to in uenza was not perturbed or enhanced in YAP/TAZ △LEC animals.In this regard, deleting these effectors did not signi cantly affect the morphological or functional characteristics measured.While Hippo signaling is involved in fundamental cellular events during both development and in tissue repair in the adult (17), our results show that YAP and TAZ do not play a role in lung LECs during the lymphangiogenic response to in uenza.In this respect, the role of Hippo signaling during lung lymphangiogenesis in adulthood contrasts with its apparent function during development.Overall, our work demonstrates that during in ammation-induced expansion of pre-existing lymphatic vasculature during adulthood, Hippo signaling is dispensable in LECs.
Broadly speaking, it is important to note that the precise role of an expanded lymphatic network accompanying tissue injury remains controversial and possibly organ and context-dependent (9,10,12,13,27,(38)(39)(40)(41).In tracheal Mycoplasma pulmonis infection, inhibiting lymphangiogenesis promotes edema and interferes with immune cell tra cking to lymph nodes (10), while in a mouse model of pulmonary brosis, promoting lymphangiogenesis was shown to reduce type I collagen accumulation (12).Further, in a mouse model of aspiration pneumonia, inhibition of lymphatic growth improved oxygen saturation (39).However, inducing lymphangiogenesis reduced in ammation, improved lymphatic drainage and increased left lung aeration in mouse lung transplant allografts (27).In the skin, lymphangiogenesis facilitates tissue resilience (9,40).Most notably, genetic blockade of lymphangiogenesis did not signi cantly affect cardiac function in a mouse model of myocardial infarction (41).Whether an expanded lymphatic network facilitates tissue recovery from diffuse lung injury will require additional study.This study demonstrates that Hippo signaling is not critical for lymphatic function and expansion during in uenza.The question as to whether the expanded network is required for recovery remains to be answered.

Mice
C57BL/6J mice were obtained from Jackson laboratories and housed in the Boston University Animal Science Center on a 12-hour light-dark cycle with access to food and water ad libitum.Prox1-Cre-ER T2 mice and TdTomato reporter mice were obtained from The Jackson Laboratory (strain #022075 and strain #007914, respectively).The YAP loxP/loxP mice and Wwtr1/TAZ loxP/loxP mice used to generate the YAP/TAZ △LEC mice reported here were provided by Dr. Jeffrey Wrana (LTRI institute) (Jackson Laboratory Strain #030532).Our study examined both male and female animals with the sex of the mice randomized across experimental groups.Similar ndings are reported for both sexes.At time of sacri ce, mice were ethically euthanized using iso urane and inferior vena cava incision.All methods were carried out in accordance with relevant IACUC guidelines and regulations and are reported in accordance with ARRIVE guidelines.All animal experiments were performed in compliance with approved Boston University animal protocols PROTO201800710_TR01 and PROTO201800057_TR01.

In uenza Infection
Mice aged 8-16 weeks were anesthetized via intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine and infected with 20-400 PFU of in uenza A/H1N1/Puerto Rico/8/34 (PR8) virus via intratracheal instillation directed into the left lung lobe.Mice were weighed daily post-infection until time of sacri ce or until all animals had returned to their pre-infection weights.In all studies, a humane endpoint of reaching 70% of starting body weight was employed.

Tamoxifen administration
To induce Cre activity, Prox1-Cre-ER T2 mice aged 6-12 weeks were administered 100 mg/kg tamoxifen dissolved in corn oil to a concentration of 30 mg/mL via intra-peritoneal injection daily for 5 days, then rested for at least 14 days before experimental use.

EdU administration
To induce EdU labeling of proliferating cells, in uenza infected or control C57BL/6J mice aged 8-12 weeks were administered 10 mg/kg EdU dissolved in sterile saline to a concentration of 2.5 mg/mL via intra-peritoneal injection daily from 4 to 6 dpi.

Tissue Processing
Mice were euthanized and their lungs perfused through the left ventricle of the heart using 10 mL of icecold HBSS.Left lungs were xed overnight at 4 o C in 4% paraformaldehyde, then washed in PBS (pH 7.4), dehydrated and embedded in para n.Para n embedded lungs were cut into 5 µm sections using a microtome and dried overnight at 42 o C.

Immunostaining and microscopy
Para n-embedded tissue sections were depara nized in xylene and then rehydrated.Antigen-retrieval was performed by heating with citrate-based antigen-unmasking solution (Vector).For experiments using HRP detection, endogenous peroxidase was quenched using 3% hydrogen peroxide in methanol.Nonspeci c antigen binding was blocked using donkey serum.Sections were incubated with primary antibodies at the dilutions shown below before application of secondary antibodies.Samples containing EdU were also counter stained per protocol using the Click-IT™ EdU Cell Proliferation Kit (Invitrogen #C10339).For HRP detection, Vectastain ABC and DAB kits were used.Sections were counterstained using hematoxylin, dehydrated and cover slipped.Samples using immuno uorescent antibodies were counter-stained with DAPI before analysis.
For PROX1 nuclei counting, non-serial murine lung tissue sections approximately 350 µm apart were stained as described above.Nuclei were visualized via light microscopy using a Nikon Eclipse E200 microscope or Leica DM4 B microscope with a Leica DFC7000 T camera.Immuno uorescent samples were imaged using a Leica DM4 B microscope with a Leica DFC7000 T camera.We would also like to acknowledge S10OD030269 instrumentation for supporting the microscopic analysis.See Table 1 for list of antibodies and dilutions.

Histopathology scoring and quantitative image analysis
Digitalized whole slide images (WSI) of bright eld H&E were generated using PhenoImager HT 2.0 Automated Quantitative Pathology Image System (Akoya Biosciences, Marlborough, Massachusetts, USA).H&E-stained slides were scanned at a 200x magni cation using a bright eld scan pro le.Quanti cation of the pulmonary in ammatory lesions were achieved by using the HALO image analysis software v3.5 (Indica labs).Each image was rst annotated by a veterinary pathologist to de ne the regions of interest for image analysis.Total examined pulmonary parenchyma with associated airways of three replicate sections were included in the region of interest.All processing artifacts (i.e.tissue folds and dust) were also manually removed via exclusion annotations.Using the random forest V2 algorithm provided with HALO image analysis software, two classes were de ned: normal and pulmonary consolidation.The full spectrum of histopathological changes was included in the algorithm, which included lymphoplasmacytic and histiocytic interstitial pneumonia and type 2 pneumocyte hyperplasia and dysplasia.The exact same classi er algorithm was applied to all images in this study to yield the pulmonary consolidation quanti cation results.The pathologist reviewed all the slides in a blinded fashion and developed ordinal histopathology criteria for scoring (Supplemental Tables 1 and 2).
For quantitative image analysis of YAP and TAZ immuno uorescent staining in LECs in lung histologic sections, PROX1 staining was used to localize the cells of interest and median uorescence intensity (MFI) of either YAP or TAZ staining was quanti ed using CellPro ler R .Data were obtained by imaging all LECs on at least 3 separate non-serial histologic lung sections per mouse.

Flow cytometry
Mice were anesthetized via intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine and injected retro-orbitally with 2 ug of IV CD45.2 antibody.This was allowed to circulate for 3 minutes prior to sacri ce.Mediastinal lymph nodes (mLNs) were collected from in uenza infected mice as well as controls and placed in 1 mL of cold PBS.LN tissue was passed through a 100 um strainer in a petri dish by crushing with a rubber syringe plunger and the strainer was washed with 3 mL cold FACS buffer.The single cell suspension was placed in 15 mL FACS tubes and centrifuged at 300xg at 4 o C for 5 minutes, and the cell pellet was resuspended in 2 mL FACS buffer.Cell number was enumerated using a hemocytometer.Cell suspensions were diluted to 1x10 6 cells/mL and 1 mL of cell suspension (1x10 6 cells) placed in new FACS tubes.Cell solutions were blocked with anti-CD16/32 (FcBlock, eBioscience) at a dilution of 1 ug per 1x10 6 cells.Fluorochrome-conjugated monoclonal antibody staining was performed using the antibodies and dilutions noted in the table for 30 minutes on ice in the dark.Excess antibody was removed by washing with 1 mL FACS buffer and centrifugation at 300xg at 4 o C for 5 minutes.The cell pellet was resuspended in 300 µL FACS buffer.Cells were stained with 7-AAD Viability Staining Solution (BD Biosci.).Unstained cells, single-stained OneComp eBeads (eBioscience), and uorescenceminus-one (FMO) controls were used for each analysis.Data were acquired on a BD LSR II ow cytometer (BD Biosciences) using BD FACSDiva software.FlowJo R 10 was utilized for data analysis.See Table 2 for list of antibodies and dilutions.

qRT-PCR
RNA isolation from mouse whole lung homogenates was performed using the Direct-zol RNA Microprep kit (Zymo Research) according to the manufacturer's instructions.qRT-PCR was performed using the Taqman and QuantStudio 3 (Applied Biosystems) systems according to the manufacturer's instructions.
See Table 3 for list of primers and probes.

Statistical analysis
Statistical signi cance was determined using a two-tailed Student's t-test in the case of data shown to be parametric via the Shapiro-Wilk test.Welches' correction was used to account for differences in variance.
If the data was non-parametric, a Mann-Whitney U test was used.A p-value of 0.05 was used as the threshold for statistical signi cance.

Figure 1
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Table 1
List of antibodies used for histologic staining.

Table 2
List of antibodies used for ow cytometry.

Table 3
(44) of primers and probes used for PCR and qRT-PCR.Mice were anesthetized via intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine before injection intratracheally into the left lung lobe with 10 µL of 10 mg/mL of 10kDa Dextran-488 (Invitrogen #D22910) dissolved in sterile normal saline (NS), or equivalent volume of NS (vehicle-only) for control mice(44).Mice were allowed to recover for 15 or 50 minutes prior to sacri ce.Mediastinal LNs and Cell debris was pelleted by centrifugation at 15,000xg at 4 o C for 20 minutes, and supernatant was collected.Fluorometric quanti cation was performed using an Agilent BioTek Synergy LX Multi-Mode uorescence plate reader.Fluorometric values for mLNs were obtained by subtracting background uorescence as measured in inguinal LNs.
(43) lung lobes were collected from in uenza infected and uninfected mice.Lungs were weighed immediately after collection to obtain the wet weight, then desiccated for 48 hours at 65 o C and weighed again to obtain the dry weight(43).