MerTK-dependent efferocytosis by monocytic-MDSCs mediates resolution of post-lung transplant ischemia-reperfusion injury

Lung transplantation (LTx) outcomes are impeded by ischemia-reperfusion injury (IRI) and subsequent chronic lung allograft dysfunction (CLAD). We examined the undefined role of MerTK (receptor Mer tyrosine kinase) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis to facilitate resolution of lung IRI. Single-cell RNA sequencing of lung tissue and bronchoalveolar lavage (BAL) from post-LTx patients were analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with Balb/c (WT), Cebpb -/- (MDSC-deficient), Mertk -/- or MerTK-CR (cleavage resistant) mice. A significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of CLAD patients was observed compared to healthy subjects. In the murine IRI model, significant increase in M-MDSCs, MerTK expression, efferocytosis and attenuation of lung dysfunction was observed in WT mice during injury resolution that was absent in Cebpb -/- and Mertk -/- mice. Adoptive transfer of M-MDSCs in Cebpb - /- mice significantly attenuated lung dysfunction and inflammation. Additionally, in a murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. Our results suggest that MerTK-dependent efferocytosis by M-MDSCs can substantially contribute to the resolution of post-LTx IRI.


INTRODUCTION
Lung transplantation (LTx) for patients with end stage lung diseases is a life-saving option that requires further investigations due to sub-optimal clinical outcomes.As post-LTx ischemiareperfusion injury (LTx-IRI) can lead to primary graft dysfunction (PGD) and chronic rejection (CLAD; chronic lung allograft dysfunction) causing the worst outcomes of all solid organ transplants, effective therapeutic modalities are urgently required to circumvent mortality in LTx recipients (1)(2)(3).IRI is characterized by immune cell infiltration and activation, increased vascular permeability, and production of inflammatory mediators, including reactive oxygen species (4).
Dysregulation of endogenous mechanisms of inflammation-resolution lead to exacerbated tissue injury and graft dysfunction (5,6).Since PGD development is a primary risk factor for CLAD, it is imperative, to understand endogenous mechanisms of inflammation-resolution that can be harnessed to facilitate graft acceptance and tissue homeostasis.
Broadly, the inflammation-resolution process is hallmarked by a variety of factors that include the presence and activation of immunosuppressive cells, the cessation of proinflammatory cell infiltration, and subsequent clearance of apoptotic cells (efferocytosis) to prevent secondary necrosis (7).Among immunosuppressive cell populations, myeloid-derived suppressor cells (MDSCs) have garnered research interest in the transplantation field both has a therapeutic measure as well as a diagnostic biomarker (8)(9)(10).This heterogenous cell population, comprising of monocytic-(M-) and granulocytic (G-or PMN-)-MDSCs are notable for their myriad of immunosuppressive/pro-resolving actions including modulation of cytokines, exhaustion of proinflammatory cells, and promotion of pro-resolving cell phenotypes (11)(12)(13)(14).A pivotal characteristic of the resolution phase is efferocytosis, which involves the clearance of apoptotic cells and debris (15).This key pro-resolving action is carried out by both professional and non-professional phagocytes at the direction of various pro-efferocytic receptors including Mer proto-oncogene tyrosine kinase (MerTK) (16,17).Although the phagocytic role of macrophages is well characterized, the contribution to the efferocytic process by infiltrating monocytic subsets, such reported that infiltrating interstitial macrophages, but not the alveolar macrophages, contributes primarily to efferocytosis of apoptotic alveolar type II epithelial cell apoptosis in lung inflammation following influenza (18).When efferocytosis is dysregulated, accumulation of apoptotic cell debris can exacerbate the inflammatory response and lead to tissue dysfunction (19).However, the role of efferocytosis and dysregulation of inflammation-resolution pathways via MerTK and infiltrating monocytic subsets in post-LTx IRI remains to be delineated.
In this study, we investigated if M-MDSCs contribute to the resolution of lung IRI via MerTK-dependent efferocytosis leading to graft survival.Our results signify the importance of dysregulation of inflammation-resolution due to MerTK-cleavage and subsequent defective efferocytosis by M-MDSCs that contributes to exacerbated tissue inflammation and post-LTx PGD.

Efferocytosis-related genes are downregulated in M-MDSCs in CLAD patients
We analyzed single-cell RNA sequencing data from CLAD patients and donor tissue (DT) from a recently reported study to identify differences in M-MDSC cell populations from myeloid cellspecific clusters (20).After data normalization and principal component analysis, we identified a total of 18 myeloid cell clusters using uniform manifold approximation and projection (UMAP) (Figure 1A).Subcluster analysis of these myeloid populations revealed Cluster 4 as M-MDSCs based on expression of HLA-DRA, ITGAM, CD33, CD14, FUT4, IL-10, VEGFA, as previously described (Supplemental Figure S1) (21,22).M-MDSCs, which display a variety of immunosuppressive capabilities, and therefore may contribute to graft tolerance were present in patients with chronic rejection, which prompted further investigation.
Analysis of differentially expressed genes (DEGs) in M-MDSCs detected 1,632 genes downregulated in CLAD patients and 308 genes downregulated in DT (Supplemental Table 1).
Since PGD can contribute to chronic rejection, we further explored differentially expressed efferocytosis-related genes (ERGs; Supplemental Table 2) in patients with CLAD, which is vital to inflammation-resolution and especially crucial in pulmonary IRI (23).In depth analysis revealed 5 of the top 30 DEGs (CD163, C25H, PELI1, IL10, VSIG4) were related to a downregulation in efferocytosis, and overall 56 differentially expressed genes were related to efferocytosis.Of the 56 ERGs, 46 were downregulated in CLAD patients (Supplemental Table 2), the majority of which were directly related to MER proto-oncogene, tyrosine kinase (MerTK)-mediated efferocytosis which includes MERTK, AXL, GAS6, ADAM9, SIRPα, CASP1, and RAC1 among others (Figure 1, B-C; Supplemental Table 3).MerTK, as a cell surface receptor is subject to cleavage, which contributes to defective efferocytosis (24).Since the cleavage of MerTK results in quantifiable soluble MER (sol-MER) in fluid secretions, we analyzed patient bronchoalveolar lavage (BAL) on days 0 and 1 post-LTx.sol-MER was significantly increased in patients on day 1 post-LTx (802.0±142.7 vs. 117.5±29.8pg/mL; p<0.0001) compared to day 0 (Figure 1D) .These clinical findings prompted us to further explore the endogenous mechanisms associated with MerTKdependent efferocytosis, via M-MDSCs, in our preclinical models of lung IRI.
To further explain the differences in PMN infiltration and activation, we investigated the processes that regulate the endogenous clearance of these cells.Both WT and Cebpb -/- demonstrated increased sol-MER levels compared to respective sham controls after IRI.sol-MER levels were significantly decreased in WT mice during resolution following IRI (24hrs) compared to inflammation phase of IRI (6hrs) (139.9±24.6 vs. 796.2±73.5 pg/mL; p<0.0001; Figure 4D).However, Cebpb -/-mice displayed significantly elevated sol-MER levels compared to WT mice at 24hrs (331.6±24 vs. 113.4±20.7 pg/mL; p=0.0004; Figure 4D).Moreover, mRNA expression of MerTK in lung tissue was significantly increased following 24hrs in WT mice compared to 6hrs (Supplemental Figure S4).Importantly, the endogenous efferocytosis by M-MDSCs in WT mice was significantly upregulated following IRI (24hrs) compared to IRI (6hrs) (46.9±1.9 vs. 15.5±3.2%; p=0.0016; Figure 4, E-F).Taken together, these data suggest that M-MDSCs are critical to the inherent resolution of lung IRI and mitigate inflammatory cytokine secretion, leukocyte trafficking, and efferocytosis.

Apoptotic PMNs undergo efferocytosis by M-MDSCs in a MerTK-dependent manner
The integral role of MerTK-dependent efferocytosis by M-MDSCs was further investigated by our in vitro studies (Figure 7A).Confocal analysis of M-MDSCs co-cultured with apoptotic PMNs demonstrated co-expression of ingested PMNs by MDSCs signifying a marked increase in efferocytosis (Figure 7B).Quantitative analysis was performed using flow cytometry which demonstrated a significant increase in uptake of apoptotic PMNs by WT M-MDSCs compared to Mertk -/-M-MDSCs (32.7±4.0 vs. 2.9±0.3%;p<0.0001) (Figure 7, C-D).Furthermore, immunosuppressive ability of M-MDSCs was evaluated by co-culture experiments using iNKT cells that are known to preferentially secrete IL-17 during lung IRI (28).The expression of IL-17 in culture supernatants was significantly increased following hypoxia-reoxygenation (HR) compared to normoxia controls, that was abrogated upon co-culture with M-MDSCs (77.0±10.8 vs. 39.4±7.2 pg/mL; p=0.001) (Supplemental Figure S6B).These results demonstrate the ability of M-MDSCs to mediate activation of key immune cells integral to lung IRI.

Resolution of lung IRI in a murine orthotopic LTx model is mediated by M-MDSCs
To further validate the findings of the hilar ligation IRI model, we assessed the role of MerTKdependent resolution of IRI in a clinically relevant murine orthotopic allogeneic LTx model (Figure 8A).Following 72hrs of post-LTx reperfusion, there was a significant increase in M-MDSC infiltration compared to 24hrs of LTx IRI (7.7±1.2 vs. 2.7±0.4 %; p=0.016) (Figure 8, B-C).This increase in M-MDSCs at 72hrs coincided with a significant decrease in albumin levels (0.3±0.04 vs. 1.0±0.06ng/mL; p=0.007; Figure 8D) signifying decrease in lung edema, as well as marked decrease in proinflammatory cytokines and increase in IL-10 expression compared to post-LTx IRI at 24hrs (Figure 8,E-G).Furthermore, enhanced efferocytosis during resolution was accompanied by a significant decrease in PMN infiltration (216.7±34.5 vs. 765.9±49.7 PMNs/HPF; p=0.0014; Figure 8, H-I), MPO expression (2301±929.5 vs. 8537±1131 pg/mL; p=0.014; Figure 8J), and sol-MER levels (283.9±44.6 vs. 667.0±110.6 pg/ml; p=0.016; Figure 8K) compared to 24hrs post-LTx IRI.Taken together, the results in both IRI models as well as in vitro studies underscore the pivotal efferocytic role of infiltrating M-MDSCs in lung tissue that immunomodulates the resolution of post-LTx IRI.

DISCUSSION
This study characterizes the importance of dysregulated resolution in PGD development which remains a significant clinical burden for LTx patients as evidenced by the 6.2-year median survival rate (29,30).The results reported herein describe a role of M-MDSCs in the resolution of lung IRI via efferocytosis of the apoptotic cell debris following post-LTx IRI.Using human LTx patient samples and two established models of lung IRI, our data delineates the mechanism of dysregulated clearance of dead cell debris that triggers uncontrolled inflammation and tissue injury, and demonstrates this process as a crucial regulator for effective resolution of lung injury and graft survival.We identified an important role of the monocytic-cell subset population that can regulate efferocytosis in two murine LTx models as well as in human LTx samples.The importance and clinical relevance of these studies is indicated by the pivotal role of MerTK receptors in mediating efferocytosis and suggesting that prevention of MerTK cleavage on monocytic immune cell populations can alleviate post-LTx IRI.
The resolution of inflammation caused by IRI, or any sterile insult, is a highly coordinated and intricate process.Reparative mechanisms for homeostatic conditions include cessation of pro-inflammatory leukocyte infiltration, efferocytosis, and the production of pro-resolving molecules (i.e.anti-inflammatory cytokines, specialized pro-resolving lipid mediators, etc.), and the presence of pro-resolving/anti-inflammatory cell populations (6,7).A particularly severe insult can disrupt one or more of these interdependent processes ultimately resulting in failed resolution.
One notable pro-resolving cell population of the inflammatory response is the immunosuppressive M-MDSC subset that has pivotal immunoregulatory capabilities, and thus, can serve as a therapeutic modality in various disease conditions (31), However the contribution of M-MDSCs in the resolution of post-LTx IRI remains to be deciphered.A recent report suggests that M-MDSC subset frequencies increase in peripheral blood after acute cellular rejection in LTx patients that could be influenced by immunosuppressive therapies (32).These findings underscore the importance of the complexity between immunosuppressive therapies, such as calcineurin or mTOR inhibition as well as dexamethasone administration in transplant patients, and endogenous immune regulation by monocytic populations such as M-MDSCs.Since the functional capabilities of inherent monocytes may be affected due to cleavage of pro-efferocytic receptors, such as TAM (Tyro3, Axl, MerTK), secondary to inflammatory milieu and/or immunosuppressive therapies, our results suggest the importance of using alternate modalities to enhance immune regulation.While research surrounding the role of MDSCs in lung IRI is largely unexplored, previous studies in other organ transplant models has investigated their potential contribution (33,34).A study of renal IRI found that depletion of both MDSC subsets via GR-1 antibody led to injury improvement and adoptive transfer of both G-and M-MDSC subsets led to worsening of renal IRI (35).Instead of using strategies that deplete or adoptively transfer all MDSC subsets, we focused on specifically using the enriched MDSC subsets for our studies to delineate their specific contribution in lung IRI.We observed that adoptive transfer of only M-MDSCs provides resolution, whereas G-MDSCs failed to provide protection in the context of lung IRI.
The immunological response during lung IRI is notably characterized by the infiltration of PMNs, which eventually undergo cell death such as apoptosis or NETosis after the initial insult has subsided (23).The clearance, or efferocytosis, of these apoptotic cells is crucial for mitigating a feedforward loop of continuing inflammation and tissue injury (15).Though this process is under regulation by a variety of receptors, MerTK is an effective efferocytic receptor due to its high level of expression in multiple tissues of the body as well as on primary phagocytes like macrophages (36).MerTK expressed on the cell surface is subject to proteolytic cleavage by ADAM17, among other molecules, resulting in the generation of soluble-Mer (sol-Mer) (37).This not only decreases cell surface function of MerTK, but sol-Mer serves as a soluble ligand that further decreases ligand-dependent interactions and activation of MerTK receptor (24).Impaired MerTK function can lead to worsening of inflammation and dysregulated repair mechanisms in atherosclerosis, bacteria-induced lung injury, and myocardial IRI, whereas prevention of MerTK cleavage with genetic or pharmacological techniques has been demonstrated to enhance inflammation-resolution (38)(39)(40).Importantly, the results in our study demonstrates the pivotal importance of preventing MerTK cleavage as a therapeutic strategy for mitigating lung injury and enhancing resolution.Moreover, since the levels of sol-Mer in BAL can act as a biomarker of the efferocytic process in the lung, an important indicator highlighted by our human patient and reciprocal murine studies, the relative contribution of MerTK-dependent efferocytosis can be potentially correlated with PGD and clinical outcomes.
In the quest for identifying therapeutic targets that can prevent MerTK cleavage, recent studies have elucidated the role of specialized proresolving lipid mediators (SPMs) such as RvD1 to prevent MerTK cleavage on macrophages and enhance efferocytosis (27,41).It is important to note that various cell types are capable of performing MerTK-dependent or -independent efferocytosis, and the relative contribution of each cell is highly dependent on the injured microenvironment.Alveolar macrophages are the primary tissue resident professional phagocyte tasked with constant surveillance.Our previous studies have also demonstrated impaired efferocytosis of alveolar macrophages during peak inflammation (42).This is likely due to MerTK cleavage on alveolar macrophages during initial insult, whereas immune trafficking subsets, such as M-MDSCs, infiltrate the site of injury with functioning MerTK and thus effectively propagate efferocytosis to facilitate resolution.Thus, the sequential contribution and role of MerTK cleavage of alveolar macrophages during early inflammation and the subsequent swarming of infiltrating monocytes like M-MDSCs during the resolution phase likely plays a crucial role in determining the fate of the lung allograft.Additionally, its plausible M-MDSCs are acting in a multifaceted manner not only for efferocytosis, but through secretion of paracrine factors (i.e.cytokines and extracellular vesicles), which remains to be further elucidated.Beyond efferocytosis, maintaining or enhancing MerTK function during acute injury has the potential to enhance anti-inflammatory mediator production, anti-inflammatory macrophage function, and regulate macrophagedependent lipid metabolism (27,(43)(44)(45).
M-MDSCs are readily recruited to sites of inflammation through canonical monocyte trafficking pathways like the CCL2/CCR2 axis (46).Depending on the inflammatory microenvironment, these cells are capable of mediating immunosuppression through secretion of anti-inflammatory cytokines, recruitment of regulatory immune cells, exhaustion of proinflammatory cells through nutrient sequestering, and facilitating cell polarization to antiinflammatory phenotypes (14).Previous studies have demonstrated the ability of MDSCs to prolong cardiac graft survival in a murine model, which was significantly reduced when MDSCs were depleted (47,48), and accordingly, M-MDSCs were shown to promote organ acceptance through recruitment of regulatory T cells in clinical kidney transplantation (49).Furthermore, studies in islet and heart transplantation have demonstrated an association between MerTK function and M-MDSC mediated transplant tolerance, mainly through their ability to recruit regulatory T cells (50).Our findings similarly suggest an upregulation of IL-10 after adoptive transfer of exogenous M-MDSCs that could be related to Tregs thereby suggesting multifaceted signaling pathways secondary to MDSC-mediated resolution of IRI.Thus, results from this study raise the interesting prospect of the role of MDSCs and preventing MerTK cleavage for enhancing immunosuppression, enhancing inflammation-resolution and alleviating post-LTx IRI.
There are limitations in this study that should be considered.The use of the hilar ligation model, which is self-resolving provides us with a high throughput way to assess the processes of resolution, but does not recapitulate the entire clinical process of LTx which includes cold preservation and donor-recipient characteristics that can further influence lung IRI.However, the use of allogeneic LTx model circumvents these concerns confirming the observed findings.
Secondly, the human translation of these experimental findings are currently limited due to the exclusion of standard clinical care immunosuppressive therapy in the preclinical murine models.
We did not use immunosuppressant therapy in the LTx model as our goal explored the inherent role of endogenous immune cell suppression by monocytic compartments without the influence of exogenous immunoregulation.However, since the standard clinical procedures involve immunosuppression in post-allograft tissue, the broad extrapolation of the M-MDSC mediated MerTK-driven immune regulation for a translational approach will require further exploration in relevantly designed future studies.Additionally, apart from MerTK, Tyro3 and Axl are a family of tyrosine kinase receptors, that may influence the process of efferocytosis and resolution (51).
These receptors may act individually or concomitantly in a disease-dependent setting and should be deciphered in subsequent studies of allograft injury (52).Also, the differentially expressed genes relative to efferocytosis in CLAD patients was not directly tested in a preclinical model of chronic rejection, as our murine models delineate PGD.However, since PGD is a primary contributor for CLAD development, analyzing efferocytosis-related gene patterns in CLAD patients provides correlative insight into mechanisms contributing to chronic allograft rejection.
Moreover, the scRNA dataset utilizes healthy donor tissue as the comparison group which may not entail donor-recipient interactions and immunosuppressant-mediated changes that can contribute to alterations in gene-expression.Therefore, to address these caveats, further investigation is required to delineate the role of impaired efferocytosis in sequential progression of PGD to CLAD in post-LTx cohorts for effective translational relevance of these preclinical findings and to decipher resolution of human lung transplant injury.In summary, our findings suggest a MerTK-mediated immunosuppressive mechanism for M-MDSCs in the resolution of lung IRI.The findings presented in this study characterize the untapped potential for the therapeutic use of M-MDSCs, as well as targeted compounds for preventing MerTK receptor cleavage, that can be enhanced ex vivo.As various cell death mechanisms have been proposed to mediate lung injury after transplant, the impending contribution of clearance pathways become of paramount importance to prevent graft rejection as well as enhance phagocytic capabilities to mitigate superimposed infections.Thus, future investigations should focus on enhancing the efferocytic ability of monocyte/macrophage populations through preservation of MerTK function for effective clinical translation in organ transplantation.

Sex as a biological variable
Human LTx analysis included both male and female patients and no sex-dimorphic effects are reported.Our study examined male and female animals, and similar findings are reported for both sexes.

Human BAL analysis
BAL collection was performed on days 0 and 1 post-LTx as routine surveillance bronchoscopy in accordance with the recommendations by International Society for Heart and Lung Transplantation consensus statement for standardization of BAL in lung transplantation (53).Two 50mL aliquots were instilled in the right middle lobe followed by aspiration.Samples were centrifuged at 500g for 5 min at 4°C and supernatants were utilized in sol-MER analysis via ELISA, per manufacturer's instructions (R&D Systems, Minneapolis, MN).

Human single cell RNA sequencing analysis
We analyzed scRNA-seq data of lungs explanted from four patients with chronic lung allograft dysfunction (CLAD) and three normal donors (DT) from published dataset GSE224210 for myeloid-cell populations and subsequent differential gene expression (20).Sequencing analysis identified 12,061 genes, of which 1,940 were differentially expressed genes (DEGs) in CLAD vs.

Lung IRI model
An established murine in vivo murine left lung hilar ligation model was used with 8-12 week old male BALB/c (wild-type; WT), C/EBPβ -/-(Cebpb -/-), C57BL/6, Mertk -/-(Jackson Laboratory), and MerTK-Cleavage Resistant (MerTK-CR; gift from Dr. Bishuang Cai, Icahn School of Medicine, Mt. Sinai, NY) mice as previously described (42,54).Briefly, animals were intubated, ventilated, and subjected to 1hr of left lung ischemia via hilar occlusion.Reperfusion was initiated upon removal of hilar suture and commenced for 6-or 24hrs, and BAL as well as left lung tissue was harvested at the end of reperfusion durations.In separate groups, mice were treated with 5.0x10 6 M-MDSCs via intravenous (i.v.) injection 24hrs prior to IRI.During both ischemia and reperfusion, mice were extubated and returned to their cage to minimize ventilator-induced injury.

Brain dead (BD) orthotopic transplantation
Murine LTx was performed on brain dead (BD) donors using C57BL/6 donor and Balb/c recipient mice for 24-or 72hrs of reperfusion, as previously described (55)(56)(57).Donors were subjected to brain death via slow inflation of a balloon catheter that was inserted via a paramedian borehole.Donor lungs were untouched for 3 hours in a period of "warm ischemia" followed by Perfadex® flush through the main pulmonary artery.Lungs were subsequently harvested and subjected to storage in 4° Perfadex® for 18hrs, and then transplanted into recipient mice.

Statistical analysis
Statistical evaluation was performed with GraphPad Prism 10 software.All values are presented as the mean ± standard error of the mean (SEM).One-way ANOVA followed by Tukey's multiple comparison test was performed to compare differences between three or more groups, and T-test followed by Mann-Whitney test.A value of P< 0.05 was considered as statistically significant.

Study Approval
Patients undergoing lung transplantation at University of Florida Health were consented for BAL fluid collection prior to transplantation in accordance with the University of Florida Institutional Review Board (#IRB201900987).All murine studies were conducted with approval from the Institutional Animal Care and Use Committee of the University of Florida under protocol #201810465.

Data availability
A Supporting Data Values file with all reported data values is available as part of the supplemental material and other supporting material are available upon request to the corresponding author.

Figure 1 .
Figure 1.Single-cell RNA-sequencing analysis of myeloid cells reveals downregulation of efferocytosis related genes in M-MDSCs of CLAD patients.(A) UMAP visualization of 18 myeloid cell clusters in lung tissue of CLAD patients and donor controls.(B) Venn diagram outlining identification of differentially expressed genes (DEGs) for MerTK-dependent efferocytosis.Downregulated genes in CLAD (blue) and donor tissue (DT; yellow) are described.(C) Volcano plot of DEGs of myeloid cell cluster 4. Genes identified by blue dots are efferocytosis-related genes with differential expression of p<0.05.Orange dots are other DEGs with p<0.05.Grey dots are genes that are not significant (p>0.05).(D) Quantification of sol-MER levels in BAL of patients showed significant increase on day 1 post-LTx compared to day 0. Data analyzed by Mann-Whitney test; *p<0.0001;n=8-10/group.

Figure 3 .
Figure 3. Pro-inflammatory cytokine expression was significantly increased in Cebpb -/-mice after IRI.(A-G) Pro-inflammatory cytokine and chemokine levels in BAL fluid were significantly

Figure 8 .Figure 1 .
Figure 8. Resolution of IRI in an allogeneic orthotopic BD model of post-LTx IRI is associated with increase in M-MDSCs.(A) Schematic depicting brain dead (BD) model of LTx in C57BL/6 donors and WT (Balb/c) recipients.(B-C) Representative flow cytometry plots and quantification of M-MDSCs in lung tissue from LTx recipients.The percentage of M-MDSCs was significantly upregulated after 72hrs of reperfusion to 24hrs.*p=0.03 vs. 24hrs; n=5-6/group.(D) Albumin levels in BAL were significantly mitigated following 72hrs of reperfusion compared to 24hrs of reperfusion.*p=0.0079;n=5/group.(E-G) Expression of pro-inflammatory cytokines in BAL was significantly mitigated and IL-10 expression was significantly increased after 72hrs compared to 24hrs of reperfusion.*p<0.04;n=5/group.(H-I) PMN infiltration in lung tissue was significantly abrogated following 72hrs of reperfusion compared to 24hrs *p=0.01;n=4-5/group.Scale bars represent 100µm.(J) MPO expression in BAL was significantly mitigated following 72hrs of reperfusion.*p=0.0079;n=5/group.(K) sol-MER levels in BAL were significantly decreased in LTx recipient mice following 72hrs of reperfusion compared to 24hrs.*p=0.016;n=5/group.Data analyzed by Mann-Whitney test.