IntroductionOur laboratory and others have shown that programmed cell death receptor-ligand 1 (PD-L1) contributes to the development of shock/sepsis-induced morbidity and mortality, but its role appears to vary across organ/cell type.ObjectiveHere, we leverage the construction of Cre-lox mouse models to produce mice constitutively lacking either PD-L1 gene expression on endothelial cells (ecPD-L1−/−) or neutrophils (pmnPD-L1−/−) to test the hypothesis that endothelial cell as opposed to neutrophil deficiency of PD-L1 differentially contributes to shock/sepsis-induced lung injury/death. Methods: Adult male C57BL/6 (breeder background strain), ecPD-L1−/−, pmnPD-L1−/−, and/or mixed flox (ec and pmn PD-L1−/+ or PD-L1+/+, respectively)-no Cre (Control) mice were subjected to either hemorrhagic (Hem) shock followed 24 h by cecal ligation and puncture (CLP) (Hem/CLP) or sham Hem and sham CLP (Sham). Survival studies were performed, and a separate set of animals were taken at 24 h post-procedure for peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue collection. Samples were processed and stained for analysis by flow cytometry, cytokine, chemokine, and angiopoietin ELISAs and indices of organ injury assays. A subset of animals was also examined for changes in lung permeability using Evan’s Blue dye exclusion.ResultsFourteen-day mortality in the ecPD-L1−/− mice was lower than in the Hem/CLP Control group, while the mortality rate was increased in the pmnPD-L1−/− vs. Controls. Lung vascular permeability was also markedly decreased in the ecPD-L1−/− Hem/CLP mice, but no such decline was seen in the lungs of pmnPD-L1−/− mice. While Hem/CLP increased the lung tissue, BALF, and blood levels of several cytokine, chemokine, and angiopoietin levels, the concentrations of lung tissue and BALF MCP-1 and blood urea nitrogen markedly declined in the ecPD-L1−/− vs. Control mice. Alternatively, the lung levels of Angiopoietin 2 and BALF MIP-2 and IL-6 concentrations significantly increased in Hem/CLP pmnPD-L1−/− animals.ConclusionsTaken together, these results support the hypothesis we have previously proffered, that expression of PD-L1 on endothelial cells has a morbid impact. However, surprisingly, we have also uncovered a potential immune protective role of PD-L1 expression on neutrophils.

Abstract Introduction Traumatic injury, like hemorrhagic shock, serves as an immune predisposition ‘priming’ event, where upon a secondary challenge a heightened inflammatory response can occur, culminating in clinical complications such as septic multiple organ failure. Large (LPM) and small (SPM) peritoneal tissue resident macrophages play a significant role in peritoneal homeostatic functions and in initiating the local immune response upon injury/infection. V-domain Ig suppressor of T-cell Activation (VISTA) has a protective effect in response to shock and septic challenge able to suppress T-cell effector function and promoting an anti-inflammatory phenotype in monocyte-derived macrophages (Mo-Macs). However, VISTA function in tissue resident peritoneal macrophages in response to shock and sepsis has yet to be studied. Methods Using a ‘double hit’ shock and sepsis induced multiple organ injury mouse model, consisting of fixed pressure hemorrhagic shock (Hem) insult followed by sepsis through the cecal ligation & puncture (CLP) method, the effects of VISTA function on the immune contribution by resident peritoneal macrophages was characterized through flow cytometry. Results Our initial data shows that following Hem/CLP, VISTA expression is upregulated in LPMs but remains constant in SPMs and Mo-Macs. However, both LPMs and SPMs are significantly depleted from the peritoneum after Hem/CLP challenge. Lastly, pro-inflammatory markers are increased in SPMs and Mo-Macs, and VISTA deficiency leads to downregulation of MHCII expression in SPMs. Conclusion These results indicate that VISTA regulates the inflammatory SPM population through its constitutive expression in peritoneal macrophages as a mechanism that promotes an anti-inflammatory phenotype and through VISTA and MHCII mediated interaction with T cells. VISTA plays a novel role as a negative regulator of innate inflammation following shock and septic insult by regulating macrophage mediated inflammation through the activation of anti-inflammatory pathways. Funding Source R35 GM118097 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Abstract Introduction Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation leading to increased microvascular permeability, pulmonary edema, and organ failure. ARDS commonly complicates septic patients, and accounts for 10% of intensive care unit admissions with a 35-45% mortality and no effective pathological-pharmacological treatment available. Bone marrow-derived macrophages (BMDMs) contribute to pulmonary damage in ARDS by adopting a pro-inflammatory phenotype and producing inflammatory mediators in the lung tissue. V-domain Ig Suppressor of T-cell Activation (VISTA), a negative checkpoint regulator expressed across the hematopoietic compartment, has been shown to promote an anti-inflammatory phenotype in macrophages and maintain a protective effect on survival in response to both septic and ARDS challenge. Methods Using an indirect ‘double hit’ ARDS mouse model of fixed pressure hemorrhagic shock (Hem) followed by a polymicrobial sepsis insult through the cecal ligation and puncture (CLP) procedure, we examined VISTA function in BMDMs from wild-type (WT) and VISTA deficient (VISTAKO) mice. Bone marrow samples were harvested from Hem/CLP challenged mice and BMDMs were cultured and polarized in-vitro for characterization through flow cytometry analysis and functional assays. Results Early data shows that VISTA KO BMDMs exhibit higher pro-inflammatory markers and MHCII expression in both Hem/CLP and sham control mice compared to WT macrophages. Lastly, VISTA KO Hem/CLP BMDMs exhibit higher production of inflammatory cytokines such as IL-6 and TNF-α even when polarized with IL-4 compared to Hem/CLP WT macrophages. Conclusion Our results highlight the role of VISTA function in BMDMs in the development of pulmonary damage seen during ARDS. Future studies will analyze how anti-inflammatory cytokine production is affected by VISTA and if VISTA agonistic treatment can alleviate increased proinflammatory function exhibited by BMDMs during ARDS. Funding Source R35 GM118097 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Introduction:Our laboratory and others have shown that Programmed cell death receptor-Ligand 1 (PD-L1), contributes to the development of shock/ sepsis induced morbidity/ mortality, but its role appears to vary across organ/cell type. Objective:Here we leverage the construction of Cre-lox mouse models to produce mice constitutively lacking either PD-L1 gene expression on endothelial cells (ecPD-L1-/-) or neutrophils (pmnPD-L1-/-), respectively, to test the hypothesis that endothelial cell as opposed to neutrophil deficiency PD-L1 differentially contributes to shock/ sepsis induced lung injury/ death. Methods:Adult male C57BL/6 (WT), ecPD-L1-/-, pmnPD-L1-/- and/or mixed flox-no cre (Control) mice were subjected to either hemorrhagic (Hem) shock followed 24 hrs by cecal ligation & puncture (CLP) (Hem/CLP) or sham Hem and sham CLP (Sham). Survival studies were done. A separate set of animals were taken at 24 hrs post-procedure for peripheral blood, broncho-alveolar lavage fluid (BALF), lung tissues were harvested, processed/ stained for flow cytometry, cytokine/ chemokine/ angiopoietin ELISAs and indices of organ injury assays. A subset of animals was also examined for changes in lung permeability using Evan's Blue dye exclusion. Results:14-day mortality in the ecPD-L1-/- mice was lower than in the Hem/CLP Control group, while the mortality rate was increased in the pmnPD-L1-/- vs. Controls. Lung vascular permeability was also markedly decreased in the ecPD-L1-/- Hem/CLP mice but no such decline was seen in the lungs of pmnPD-L1-/- mice. While Hem/CLP increased the lung tissue, BALF and blood levels of several cytokine/ chemokine/angiopoietin levels, the concentrations of lung tissue, BALF MCP-1 and blood BUN markedly declined in the ecPD-L1-/- vs. Control mice. Alternatively, the lung levels of Angiopoietin-2 and BALF MIP-2 and IL-6 concentrations significantly increased in Hem/CLP pmnPD-L1-/- animals. Conclusions:Taken together, these results support the hypothesis we have previously proffered that expression of PD-L1 on endothelial cells has a morbid impact. However, surprisingly, we have also uncovered a potential immune protective role of PD-L1 expression on neutrophils.

Acute respiratory distress syndrome (ARDS), a common complication seen in septic patients, is characterized by lung epithelial and endothelial barrier dysfunction leading to pulmonary edema and organ failure. Currently, ARDS has a 30-40% mortality rate with no effective pathological-pharmacological based treatment available. Bone marrow-derived macrophages (BMDMs) contribute to the immune dysfunction and organ damage experienced in ARDS patients when they are recruited into affected tissues and adapt a pro-inflammatory phenotype. The negative checkpoint regulator V-domain Ig suppressor of T-cell Activation (VISTA) from the B7/CD28 superfamily is expressed across the hematopoietic compartment and has been shown to promote an anti-inflammatory protective effect on survival in sepsis mouse models. Using an established indirect ARDS animal model, characterized by an initial insult of fixed pressure hemorrhagic shock followed by polymicrobial sepsis through cecal ligation puncture, the therapeutic potential of VISTA treatment was examined. Bone marrow samples from iARDS mice were harvested and VISTA expression and function from polarized BMDMs was characterized with flow cytometry and functional assays. Preliminary data has revealed an impaired reduction of IL-6 in iARDS BMDMs polarized with IL-4 when compared to their sham controls. Our results examine the anti-inflammatory function mediated by VISTA in monocyte-derived macrophages as a potential treatment of septic ARDS. Supported by NIH R35 GM118097 Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Background:Indirect acute respiratory distress syndrome (iARDS) is a life-threatening inflammatory lung injury often triggered by extrapulmonary insults. Although immune checkpoints are critical regulators of inflammation, the role of V-domain Ig suppressor of T-cell activation (VISTA) in iARDS remains unexplored. Methods:Using a murine model of iARDS, we compared outcomes in VISTA knockout (VISTA-/-) and wild-type mice. Disease severity was assessed through lung injury scoring, survival analysis, and cytokine/chemokine profiling in plasma, lung tissue, and peritoneal fluid. The therapeutic potential of VISTA was evaluated using an anti-VISTA antibody (13F3). Results:VISTA-/- mice exhibited exacerbated lung injury, reduced survival, and elevated systemic levels of interleukin (IL)-6, IL-10, MIP-2, and KC compared to wild-type controls. While cytokine levels in lung tissue remained stable, peritoneal fluid mediators were dysregulated in VISTA-/- mice, highlighting compartment-specific inflammatory regulation. Treatment with 13F3 reduced VISTA expression on myeloid and structural cells (monocytes, neutrophils, macrophages, epithelium, endothelium) and partially modulated cytokine/chemokine profiles across compartments. Conclusion:Our findings establish VISTA as a protective immune checkpoint in iARDS that restrains systemic hyperinflammation and organ damage. Although antibody-mediated VISTA targeting altered inflammatory pathways, its incomplete efficacy suggests complex, multifactorial mechanisms at play. These results position VISTA as a novel therapeutic target for iARDS and warrant further exploration of timed immunomodulatory strategies to harness its protective effects.

INTRODUCTION:Acute respiratory distress syndrome (ARDS) is a life-threatening pulmonary condition with significant mortality, largely due to a lack of therapeutic interventions grounded in its molecular pathophysiology. Immune checkpoint regulators, such as the V-domain Ig Suppressor of T cell Activation (VISTA), may provide novel immunotherapeutic strategies for ARDS by modulating the immune response, a concept extensively explored in cancer and autoimmune diseases. Investigating VISTA in the context of ARDS could unveil new therapeutic avenues. METHODS:We used a mouse model of indirect ARDS by subjecting C57BL/6J mice to hemorrhage followed by cecal ligation and puncture. Systemic and localized inflammatory conditions were assessed using samples from blood, lung, and peritoneal fluid. Lung pathology was quantified by scoring hematoxylin and eosin-stained sections. Flow cytometry, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction analyses concentrated on macrophages, neutrophils, endothelial cells, and epithelial cells to elucidate VISTA expression patterns. RESULTS:Hemorrhage or cecal ligation and puncture-treated mice exhibited hallmark symptoms of indirect ARDS, including elevated levels of inflammatory cytokines and chemokines. Notably, VISTA expression was substantially upregulated on various cell types, including blood monocytes, lung macrophages, and both circulating and lung-infiltrating neutrophils, as well as on pulmonary epithelial cells and endothelial cells. CONCLUSIONS:Our model replicates critical inflammatory and physiologic changes leading to ARDS, with the elevated expression of VISTA on immune and parenchymal cells suggesting its central involvement in lung injury. The findings propose VISTA as both a potential biomarker for lung damage and as a promising target for ARDS therapy.

Sepsis is a dysregulated systemic immune response to infection i.e. responsible for similar to 35% of in-hospital deaths at a significant fiscal healthcare cost. Our laboratory, among others, has demonstrated the efficacy of targeting negative checkpoint regulators (NCRs) to improve survival in a murine model of sepsis, cecal ligation and puncture (CLP). B7-CD28 superfamily member, V-domain immunoglobulin suppressor of T cell activation (VISTA), is an ideal candidate for strategic targeting in sepsis. VISTA is a 35 to 45 kDa type 1 transmembrane protein with unique biology that sets it apart from all other NCRs. We recently reported that VISTA(-/-) mice had a significant survival deficit post-CLP, which was rescued upon adoptive transfer of a VISTA-expressing pMSCV-mouse Foxp3-EF1 alpha-GFP-T2A-puro stable Jurkat cell line (Jurkat(foxp3) T cells). Based on our prior study, we investigated the effector cell target of Jurkat(foxp3) T cells in VISTA(-/-) mice. gamma delta T cells are a powerful lymphoid subpopulation that require regulatory fine-tuning by regulatory T cells to prevent overt inflammation/pathology. In this study, we hypothesized that Jurkat(foxp3) T cells nonredundantly modulate the gamma delta T cell population post-CLP. We found that VISTA(-/-) mice have an increased accumulation of intestinal CD69(low) gamma delta T cells, which are not protective in murine sepsis. Adoptive transfer of Jurkat(foxp3) T cells decreased the intestinal gamma delta T cell population, suppressed proliferation, skewed remaining gamma delta T cells toward a CD69(high) phenotype, and increased soluble CD40L in VISTA(-/-) mice post-CLP. These results support a potential regulatory mechanism by which VISTA skews intestinal gamma delta T cell lineage representation in murine sepsis.

IntroductionAcute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a commonly occurring sequelae of traumatic injury resulting from indirect insults like hypovolemic shock and/or extrapulmonary sepsis. The high lethality rate associated with these pathologies outlines the importance of clarifying the “priming” effects seen in the post-shock lung microenvironment, which are understood to bring about a dysregulated or overt immune response when triggered by a secondary systemic infectious/septic challenge culminating in ALI. In this pilot project, we test the hypothesis that application of a single cell multiomics approach can elucidate novel phenotype specific pathways potentially contributing to shock-induced ALI/ARDS.MethodsHypovolemic shock was induced in C57BL/6 (wild-type), PD-1, PD-L1, or VISTA gene deficient male mice, 8–12 weeks old. Wild-type sham surgeries function as negative controls. A total of 24-h post-shock rodents were sacrificed, their lungs harvested and sectioned, with pools prepared from 2 mice per background, and flash frozen on liquid nitrogen. N = 2 biological replicates (representing 4 mice total) were achieved for all treatment groups across genetic backgrounds. Samples were received by the Boas Center for Genomics and Human Genetics, where single cell multiomics libraries were prepared for RNA/ATAC sequencing. The analysis pipeline Cell Ranger ARC was implemented to attain feature linkage assessments across genes of interest.ResultsSham (pre-shock) results suggest high chromatin accessibility around calcitonin receptor like receptor (CALCRL) across cellular phenotypes with 17 and 18 feature links, exhibiting positive correlation with gene expression between biological replicates. Similarity between both sample chromatin profiles/linkage arcs is evident. Post-shock wild-type accessibility is starkly reduced across replicates where the number of feature links drops to 1 and 3, again presenting similar replicate profiles. Samples from shocked gene deficient backgrounds displayed high accessibility and similar profiles to the pre-shock lung microenvironment.ConclusionHigh pre-shock availability of DNA segments and their positive correlation with CALCRL gene expression suggests an apparent regulatory capacity on transcription. Post-shock gene deficient chromatin profiles presented similar results to that of pre-shock wild-type samples, suggesting an influence on CALCRL accessibility. Key changes illustrated in the pre-ALI context of shock may allow for additional resolution of “priming” and “cellular pre-activation/pre-disposition” processes within the lung microenvironment.

Sepsis is a systemic immune response to infection that is responsible for ~35% of in-hospital deaths and over 24 billion dollars in annual treatment costs. Strategic targeting of non-redundant negative immune checkpoint protein pathways can cater therapeutics to the individual septic patient and improve prognosis. B7-CD28 superfamily member V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an ideal candidate for strategic targeting in sepsis. We hypothesized that immune checkpoint regulator, VISTA, controls T-regulatory cells (Treg), in response to septic challenge, thus playing a protective role/reducing septic morbidity/mortality. Further, we investigated if changes in morbidity/mortality are due to a Treg-mediated effect during the acute response to septic challenge. To test this, we used the cecal ligation and puncture model as a proxy for polymicrobial sepsis and assessed the phenotype of CD4+ Tregs in VISTA-gene deficient (VISTA-/-) and wild-type mice. We also measured changes in survival, soluble indices of tissue injury, and circulating cytokines in the VISTA-/- and wild-type mice. We found that in wild-type mice, CD4+ Tregs exhibit a significant upregulation of VISTA which correlates with higher Treg abundance in the spleen and small intestine following septic insult. However, VISTA-/- mice have reduced Treg abundance in these compartments met with a higher expression of Foxp3, CTLA4, and CD25 compared to wild-type mice. VISTA-/- mice also have a significant survival deficit, higher levels of soluble indicators of liver injury (i.e., ALT, AST, bilirubin), and increased circulating proinflammatory cytokines (i.e., IL-6, IL-10, TNFα, IL-17F, IL-23, and MCP-1) following septic challenge. To elucidate the role of Tregs in VISTA-/- sepsis mortality, we adoptively transferred VISTA-expressing Tregs into VISTA-/- mice. This adoptive transfer rescued VISTA-/- survival to wild-type levels. Taken together, we propose a protective Treg-mediated role for VISTA by which inflammation-induced tissue injury is suppressed and improves survival in early-stage murine sepsis. Thus, enhancing VISTA expression or adoptively transferring VISTA+ Tregs in early-stage sepsis may provide a novel therapeutic approach to ameliorate inflammation-induced death.

Morbidity and mortality associated with neonatal sepsis remains a healthcare crisis. PD1 −/− neonatal mice endured experimental sepsis, in the form of cecal slurry (CS), and showed improved rates of survival compared to wildtype (WT) counterparts. End-organ injury, particularly of the lung, contributes to the devastation set forth by neonatal sepsis. PDL1 −/− neonatal mice, in contrast to PD1 −/− neonatal mice did not have a significant improvement in survival after CS. Because of this, we focused subsequent studies on the impact of PD1 gene deficiency on lung injury. Here, we observed that at 24 h post-CS (but not at 4 or 12 h) there was a marked increase in pulmonary edema (PE), neutrophil influx, myeloperoxidase (MPO) levels, and cytokine expression sham (Sh) WT mice. Regarding pulmonary endothelial cell (EC) adhesion molecule expression, we observed that Zona occludens-1 (ZO-1) within the cell shifted from a membranous location to a peri-nuclear location after CS in WT murine cultured ECs at 24hrs, but remained membranous among PD1 −/− lungs. To expand the scope of this inquiry, we investigated human neonatal lung tissue. We observed that the lungs of human newborns exposed to intrauterine infection had significantly higher numbers of PD1 + cells compared to specimens who died from non-infectious causes. Together, these data suggest that PD1/PDL1, a pathway typically thought to govern adaptive immune processes in adult animals, can modulate the largely innate neonatal pulmonary immune response to experimental septic insult. The potential future significance of this area of study includes that PD1/PDL1 checkpoint proteins may be viable therapeutic targets in the septic neonate.

Background: Hypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic setting. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major protein phosphatase tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to development of AKI sequential hemorrhage (Hem) and cecal ligation and puncture (CLP) and whether inactivation of SHP2 through administration of its selective inhibitor, phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury. Methods: Male C57BL/6 mice were subjected to Hem (a “priming” insult) followed by CLP or sham-Hem plus sham-CLP (S/S) as controls. Samples of blood and kidney were harvested at 24 h post CLP. The expression of neutrophil gelatinase-associated lipocalin (NGAL) , high mobility group box 1 ( HMGB1 ), caspase3 as well as SHP2:phospho-SHP2, extracellular-regulated kinase (Erk1/2) :phospho-Erk1/2, and signal transducer and activator of transcription 3 (STAT3):phospho-STAT3 protein in kidney tissues were detected by Western blotting. The levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum were measured according to the manufacturer’s instructions. Blood inflammatory cytokine/chemokine levels were detected by ELISA. Results: We found that indices of kidney injury, including levels of BUN, Cre and NGAL as well as histopathologic changes, were significantly increased after Hem/CLP in comparison with that in the S/S group. Furthermore, Hem/CLP resulted in elevated serum levels of inflammatory cytokines/chemokines, and induced increased levels of HMGB1, SHP2:phospho-SHP2, Erk1/2:phospho-Erk1/2, and STAT3:phospho-STAT3 protein expression in the kidney. Treatment with PHPS1 markedly attenuated these Hem/CLP-induced changes. Conclusions: In conclusion, our data indicate that SHP2 inhibition attenuates AKI induced by our double-hit/sequential insult model of Hem/CLP and that this protective action may be attributable to its ability to mitigate activation of the Erk1/2 and STAT3 signaling pathway. We believethis is a potentially important finding with clinical implications warranting further investigation.

Background Hypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic course. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major phosphatase protein tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to AKI induced by hemorrhage (Hem) followed by cecal ligation and puncture (CLP) (Hem/CLP) and, further, if inactivation of SHP2 with the selective inhibitor, Phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury. Methods Male C57BL/6 mice were subjected to Hem (a “priming” insult) followed by CLP or sham-Hem plus sham-CLP (S/S) as controls. Samples of blood and kidney were harvested at 24 h post CLP. The expression of neutrophil gelatinase-associated lipocalin (NGAL), high mobility group box 1 (HMGB1), caspase3 as well as SHP2:phospho-SHP2, extracellular-regulated kinase (Erk1/2):phospho-Erk1/2, and signal transducer and activator of transcription 3 (STAT3):phospho-STAT3 protein in kidney tissues were detected by Western blotting. The levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum were measured according to the manufacturer’s instructions. Blood inflammatory cytokine/chemokine levels were detected by ELISA. Results Here we report that indices of kidney injury, such as BUN, Cre, NGAL in renal tissue and histopathologic change, were pronounced after Hem/CLP in comparison with that observed in the S/S group mice. Furthermore, while Hem/CLP elevated serum levels of inflammatory cytokine/ chemokine, and induced increased levels of HMGB1, SHP2:phospho-SHP2, Erk1/2:phospho-Erk1/2, as well asSTAT3:phospho-STAT3 protein expression in the kidney; treatment with PHPS1 markedly attenuated these Hem/CLP induced changes. Conclusions In conclusion, our data indicate that SHP2 inhibition attenuates AKI induced by our double-hit/sequential insult model of Hem/CLP and that this protective action may be attributable to its anti-inflammatory property in mitigating activation of the Erk1/2 and STAT3 signaling pathway. This finding we believe has important potential clinical implications and, thus, warrants further investigation.

This study sets out to establish the comparative contribution of PD-L1 expression by pulmonary endothelial cells (ECs) and/or epithelial cells (EpiCs) to the development of indirect acute lung injury (iALI) by taking advantage of the observation that treatment with naked siRNA by intratracheal delivery in mice primarily affects lung EpiCs, but not lung ECs, while intravenous delivery of liposomal-encapsulated siRNA largely targets vascular ECs including the lung, but not pulmonary EpiCs. We showed that using a mouse model of iALI [induced by hemorrhagic shock followed by septic challenge (Hem-CLP)], PD-L1 expression on pulmonary ECs or EpiCs was significantly upregulated in the iALI mice at 24 h post-septic insult. After documenting the selective ability of intratracheal versus intravenous delivery of PD-L1 siRNA to inhibit PD-L1 expression on EpiCs versus ECs, respectively, we observed that the iALI-induced elevation of cytokine/chemokine levels (in the bronchoalveolar lavage fluid, lung lysates, or plasma), lung myeloperoxidase and caspase-3 activities could largely only be inhibited by intravenous, but not intratracheal, delivery of PD-L1 siRNA. Moreover, intravenous, but not intratracheal, delivery led to a preservation of normal tissue architecture, lessened pulmonary edema, and reduced neutrophils influx induced by iALI. In addition, in vitro mouse endothelial cell line studies showed that PD-L1 gene knockdown by siRNA or knockout by CRISPR/Cas9-mediated gene manipulation, reduced monolayer permeability, and maintained tight junction protein levels upon recombinant IFN-γ stimulation. Together, these data imply a critical role for pulmonary vascular ECs in mediating PD-1:PD-L1-driven pathological changes resulting from systemic stimuli such as Hem-CLP.

Therapeutic interventions to treat acute lung injury (ALI) remain largely limited to lung-protective strategies, as a real molecular pathophysiologically driven therapeutic intervention has yet to become available. While we have previously documented the expression of herpes virus entry mediator (HVEM) on leukocytes of septic mice and critically ill patients, its functional role in shock/sepsis-induced ALI has not yet been studied. Inasmuch, a murine model of indirect ALI (iALI) was induced by hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), septic challenge and HVEM-siRNA or phosphate buffered saline was administrated by intratracheal instillation 2 h after hemorrhage to determine the role of HVEM in the development of experimental iALI. Indices of lung injury were measured. HVEM expression was significantly elevated in iALI mice. Compared with phosphate buffered saline treated iALI mice, HVEM knock-down by siRNA caused a reduction of cytokine/chemokine levels, myeloperoxidase activity, broncho-alveolar lavage fluid (BALF) cell count and protein concentration. HVEM-siRNA treatment reduced inflammation and attenuated pulmonary architecture destruction as well as provided an early (60 h post HEM-CLP) survival benefit in iALI mice. This ability of anti-HVEM treatment to prevent the development of iALI and provide a transient survival benefit implies that mitigating signaling through HVEM may be a novel target worth further investigation.