Persistent Inflammation, Immunosuppression, and Catabolism Syndrome in Pediatric Populations: A Brief Perspective.

Surviving near-lethal insults, such as sepsis, trauma, and major surgery is more common due to advances in medical care. The decline in mortality has unmasked a population of chronic critically ill patients, many with the pathological immunophenotype known as Persistent inflammation, Immunosuppression, and Catabolism Syndrome (PICS). Though initially described in adults, many critically ill children exhibit the hallmarks of PICS, including lymphopenia, hyperinflammation, and evidence of ongoing somatic protein catabolism. These patients are plagued with recurrent infections and suffer worse outcomes. There remains a need to understand the pathophysiology underlying this condition to elucidate potential therapies and develop interventions. This perspective provides the most current update of PICS within the pediatric population.

DIFFERENTIAL SIGNALING EFFECTS OF ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS IN HUMAN WHOLE BLOOD INDICATE DISTINCT REGULATION OF THE NRF2 PATHWAY.

ABSTRACT: Escherichia coli and Staphylococcus aureus are two of the most common bacterial species responsible for sepsis. While it is observed that they have disparate clinical phenotypes, the signaling differences elicited by each bacteria that drive this variance remain unclear. Therefore, we used human whole blood exposed to heat-killed E. coli or S. aureus and measured the transcriptomic signatures. Relative to unstimulated control blood, heat-killed bacteria exposure led to significant dysregulation (upregulated and downregulated) of >5,000 genes for each experimental condition, with a slight increase in gene alterations by S. aureus. While there was significant overlap regarding proinflammatory pathways, Gene Ontology overrepresentation analysis of the most altered genes suggested biological processes like macrophage differentiation and ubiquinone biosynthesis were more unique to heat-killed S. aureus, compared with heat-killed E. coli exposure. Using Ingenuity Pathway Analysis, it was demonstrated that nuclear factor erythroid 2-related factor 2 signaling, a main transcription factor in antioxidant responses, was predominately upregulated in S. aureus exposed blood relative to E. coli. Furthermore, the use of pharmacologics that preferentially targeted the nuclear factor erythroid 2-related factor 2 pathway led to differential cytokine profiles depending on the type of bacterial exposure. These findings reveal significant inflammatory dysregulation between E. coli and S. aureus and provide insight into the targeting of unique pathways to curb bacteria-specific responses.

Vascular Inflammation and Smooth Muscle Contractility: The Role of Nox1-Derived Superoxide and LRRC8 Anion Channels.

Vascular inflammation underlies the development of hypertension, and the mechanisms by which it increases blood pressure remain the topic of intense investigation. Proinflammatory factors including glucose, salt, vasoconstrictors, cytokines, wall stress, and growth factors enhance contractility and impair relaxation of vascular smooth muscle cells. These pathways share a dependence upon redox signaling, and excessive activation promotes oxidative stress that promotes vascular aging. Vascular smooth muscle cell phenotypic switching and migration into the intima contribute to atherosclerosis, while hypercontractility increases systemic vascular resistance and vasospasm that can trigger ischemia. Here, we review factors that drive the initiation and progression of this vasculopathy in vascular smooth muscle cells. Emphasis is placed on the contribution of reactive oxygen species generated by the Nox1 NADPH oxidase which produces extracellular superoxide (O2•-). The mechanisms of O2•- signaling remain poorly defined, but recent evidence demonstrates physical association of Nox1 with leucine-rich repeat containing 8 family volume-sensitive anion channels. These may provide a pathway for influx of O2•- to the cytoplasm, creating an oxidized cytoplasmic nanodomain where redox-based signals can affect both cytoskeletal structure and vasomotor function. Understanding the mechanistic links between inflammation, O2•- and vascular smooth muscle cell contractility may facilitate targeting of anti-inflammatory therapy in hypertension.

Extracellular SOD modulates canonical TNFα signaling and α5ß1 integrin transactivation in vascular smooth muscle cells.

TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3ß (GSK3ß), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5ß1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5ß1 integrin activation.

Protein-mediated interactions in the dynamic regulation of acute inflammation.

Protein-mediated interactions are the fundamental mechanism through which cells regulate health and disease. These interactions require physical contact between proteins and their respective targets of interest. These targets include not only other proteins but also nucleic acids and other important molecules as well. These proteins are often involved in multibody complexes that work dynamically to regulate cellular health and function. Various techniques have been adapted to study these important interactions, such as affinity-based assays, mass spectrometry, and fluorescent detection. The application of these techniques has led to a greater understanding of how protein interactions are responsible for both the instigation and resolution of acute inflammatory diseases. These pursuits aim to provide opportunities to target specific protein interactions to alleviate acute inflammation.

LRRC8A anion channels modulate vascular reactivity via association with myosin phosphatase rho interacting protein.

Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.

Pediatric Persistent Inflammation, Immunosuppression, and Catabolism Syndrome Prevalence in Sepsis-Related Mortalities: A 23-Year Institutional History.

BACKGROUND: Delayed mortality in sepsis often is linked to a lack of resolution in the inflammatory cascade termed persistent inflammation, immunosuppression, and catabolism syndrome (PICS). Limited research exists on PICS in pediatric patients with sepsis. RESEARCH QUESTION: What is the prevalence of pediatric PICS (pPICS) in patients who died of sepsis-related causes and what associated pathogen profiles and comorbidities did they have compared with those patients without pPICS who died from sepsis? STUDY DESIGN AND METHODS: A retrospective study of a single institution using a de-identified database from 1997 through 2020 for all patients aged 21 years or younger who died of culture-positive sepsis from a known source and who had laboratory data available were evaluated for the presence of pPICS. RESULTS: Among records extracted from the institutional database, 557 patients had culture-positive sepsis, with 262 patients having pPICS (47%). Patients with pPICS were more likely to have underlying hematologic or oncologic disease or cardiac disease. In addition, patients who had pPICS showed increased odds of associated fungal infection compared with those patients who did not (OR, 2.69; 95% CI, 1.59-4.61; P < .001). When assessing laboratory criteria, having a sustained absolute lymphocyte count of < 1.0 × 103/µL was most closely associated with having pPICS compared with other laboratory parameters. Finally, the results of multivariate logistic regression analysis indicated that patients with pPICS were more common in the cardiac ICU, as opposed to the PICU (OR, 3.43; CI, 1.57-7.64; P = .002). INTERPRETATION: Pediatric patients who died of a sepsis-related cause have a pPICS phenotype nearly one-half of the time. These patients are more likely to be in the cardiac ICU than the pediatric ICU and have associated fungal infections. Special attention should be directed toward this population in future research.

LRRC8A anion channels modulate vasodilation via association with Myosin Phosphatase Rho Interacting Protein (MPRIP).

Background: In vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O 2 -• ). Methods and Results: Mice lacking LRRC8A exclusively in VSMCs (Sm22α-Cre, KO) were used to assess the role of VRACs in TNFα signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours of ex vivo exposure to TNFα (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Conclusions: Interaction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.

Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.

Chloride channel-3 (ClC-3) Cl-/H+ antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A, and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labeled vesicles were sorted to MVBs labeled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.

Case report: Temporal alterations in vascular function during the first 2 weeks of pediatric septic shock.

Introduction: During sepsis and septic shock, the host's immune systems generate an overwhelming and often, detrimental, inflammatory response. Part of this response results in significant alterations in blood flow and vasomotor tone regulated in part by endothelial and vascular smooth muscle cells. Here, we report on a series of 3 pediatric patients for whom vascular response was assessed by laser doppler perfusion coupled to iontophoresis over the first 2 weeks after hospitalization for septic shock to demonstrate similarities and dissimilarities in the vascular response. Case Presentations: A 12-year-old male with a history of Burkitt's Lymphoma, a 21-year-old male with congenital porencephaly and epilepsy, and a 7-year-old male with no significant past medical history all were admitted to a tertiary care children's hospital with a diagnosis of septic shock requiring vasoactive infusions to maintain mean arterial blood pressure. Non-invasive laser doppler perfusion coupled with iontophoresis of either acetylcholine (endothelial-dependent response) or sodium nitroprusside (endothelial-independent response) was performed on hospital days 1, 3, 7, and 14. Variability and heterogeneity were demonstrated by the temporal assessments of the vascular response to sodium nitroprusside, but all three patients showed significant similarity in the temporal responsiveness to acetylcholine. Conclusion: Assessment of baseline and temporal responsiveness to endothelial-dependent vascular reactivity may provide a predictable timeline to the resolution of pediatric septic shock.

Endothelial-Dependent Responses Correlate with Pediatric SOFA Scores During Severe Sepsis and Septic Shock.

Sepsis is an exaggerated host response to an infectious challenge that is associated with significant alterations in vasomotor tone. We hypothesized that the endothelial dysfunction observed during severe sepsis and septic shock would correlate with the degree of organ failure as determined by the pediatric Sequential Organ Failure Assessment (pSOFA) score. Utilizing laser Doppler perfusion monitoring coupled with iontophoresis, we found that endothelium-dependent vascular reactivity to acetylcholine (ACh) stimulation significantly correlated with both total pSOFA scores and, more specifically, cardiovascular (CV) pSOFA scores. Alternatively, endothelium-independent vascular reactivity using sodium nitroprusside (SNP) did not demonstrate a significant relationship with pSOFA scores. These data suggest that endothelial-mediated vasculopathy may be a key driver of organ dysfunction during episodes of pediatric sepsis.

Cell penetrating peptides coupled to an endothelial nitric oxide synthase sequence alter endothelial permeability.

Delivery of cargo to cells through the use of cell-penetrating peptide (CPP) sequences is an area of rich investigation for targeted therapeutics. Specific to the endothelium, the layer of cells that cover every blood vessel in the body, the loss or alteration of a key enzyme, endothelial nitric oxide synthase (eNOS), is known to contribute to endothelial health during severe, infectious challenge. While the beneficial effects of eNOS are often thought to be mediated through the generation of nitric oxide, some protection is theorized to be through eNOS binding to regulatory pathways via a pentabasic RRKRK motif. We hypothesized that delivery of the eNOS-RRKRK peptide sequence using common CPPs would allow protection against gram-negative lipopolysaccharide (LPS). Combination of the eNOS-RRKRK sequence to the CPP antennapedia (AP) reduced the impact of LPS-induced permeability in cultured human microvascular endothelial cells (HMVECs) as measured by transendothelial electrical resistance (TEER). There was also a modest reduction in cytokine production, however it was observed that AP alone significantly impaired LPS-induced endothelial permeability and cytokine production. In comparison, the CPP trans-activator of transcription (TAT) did not significantly alter endothelial inflammation by itself. When TAT was coupled to the eNOS-RRKRK sequence, protection against LPS-induced permeability was still demonstrated, however cytokine production was not reduced. These data demonstrate that the RRKRK sequence of eNOS can offer some NO-independent protection against LPS-mediated endothelial inflammation, however the degree of protection is highly dependent on the type of CPP utilized for cargo delivery.

Apoptosis signal-regulating kinase 1 (ASK1) inhibition reduces endothelial cytokine production without improving permeability after toll-like receptor 4 (TLR4) challenge.

Sepsis represents a life-threatening event often mediated by the host's response to pathogens such as gram-negative organisms, which release the proinflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.

The Inverse Relationship Between Endothelium-Dependent Vasodilation and Blood Pressure is Lost After Cardiopulmonary Bypass.

Cardiopulmonary bypass (CPB) is required for the surgical correction of congenital heart defects and incites an acute inflammatory response that impairs endothelial function post-operatively. Therefore, we hypothesized that the pre-operative relationship between endothelial function and blood pressure would be impaired after CPB-mediated inflammation. Using laser Doppler perfusion monitoring coupled with iontophoresis, we found that while there was a significant inverse correlation between endothelium-dependent vascular reactivity to acetylcholine (ACh) stimulation and systolic blood pressure (SBP), this relationship was lost after CPB. No relationship was observed between endothelium-independent vascular reactivity using sodium nitroprusside (SNP) and SBP either pre-CPB or any point thereafter. Additionally, neither CPB time nor inflammatory cytokines correlated with the degree of responsiveness to ACh. These data suggest that the measurement of endothelium impairment after CPB may be more reflective of cardiovascular health than SBP alone.

TNFα and Reactive Oxygen Signaling in Vascular Smooth Muscle Cells in Hypertension and Atherosclerosis.

Hypertension and atherosclerosis, the predecessors of stroke and myocardial infarction, are chronic vascular inflammatory reactions. Tumor necrosis factor alpha (TNFα), the "master" proinflammatory cytokine, contributes to both the initiation and maintenance of vascular inflammation. TNFα induces reactive oxygen species (ROS) production which drives the redox reactions that constitute "ROS signaling." However, these ROS may also cause oxidative stress which contributes to vascular dysfunction. Mice lacking TNFα or its receptors are protected against both acute and chronic cardiovascular injury. Humans suffering from TNFα-driven inflammatory conditions such as rheumatoid arthritis and psoriasis are at increased cardiovascular risk. When treated with highly specific biologic agents that target TNFα signaling (Etanercept, etc.) they display marked reductions in that risk. The ability of TNFα to induce endothelial dysfunction, often the first step in a progression toward serious vasculopathy, is well recognized and has been reviewed elsewhere. However, TNFα also has profound effects on vascular smooth muscle cells (VSMCs) including a fundamental change from a contractile to a secretory phenotype. This "phenotypic switching" promotes proliferation and production of extracellular matrix proteins which are associated with medial hypertrophy. Additionally, it promotes lipid storage and enhanced motility, changes that support the contribution of VSMCs to neointima and atherosclerotic plaque formation. This review focuses on the role of TNFα in driving the inflammatory changes in VSMC biology that contribute to cardiovascular disease. Special attention is given to the mechanisms by which TNFα promotes ROS production at specific subcellular locations, and the contribution of these ROS to TNFα signaling.

Endothelial-Dependent Vasomotor Dysfunction in Infants After Cardiopulmonary Bypass.

OBJECTIVES: Cardiopulmonary bypass-induced endothelial dysfunction has been inferred by changes in pulmonary vascular resistance, alterations in circulating biomarkers, and postoperative capillary leak. Endothelial-dependent vasomotor dysfunction of the systemic vasculature has never been quantified in this setting. The objective of the present study was to quantify acute effects of cardiopulmonary bypass on endothelial vasomotor control and attempt to correlate these effects with postoperative cytokines, tissue edema, and clinical outcomes in infants. DESIGN: Single-center prospective observational cohort pilot study. SETTING: Pediatric cardiac ICU at a tertiary children's hospital. PATIENTS: Children less than 1 year old requiring cardiopulmonary bypass for repair of a congenital heart lesion. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Laser Doppler perfusion monitoring was coupled with local iontophoresis of acetylcholine (endothelium-dependent vasodilator) or sodium nitroprusside (endothelium-independent vasodilator) to quantify endothelial-dependent vasomotor function in the cutaneous microcirculation. Measurements were obtained preoperatively, 2-4 hours, and 24 hours after separation from cardiopulmonary bypass. Fifteen patients completed all laser Doppler perfusion monitor (Perimed, Järfälla, Sweden) measurements. Comparing prebypass with 2-4 hours postbypass responses, there was a decrease in both peak perfusion (p = 0.0006) and area under the dose-response curve (p = 0.005) following acetylcholine, but no change in responses to sodium nitroprusside. Twenty-four hours after bypass responsiveness to acetylcholine improved, but typically remained depressed from baseline. Conserved endothelial function was associated with higher urine output during the first 48 postoperative hours (R = 0.43; p = 0.008). CONCLUSIONS: Cutaneous endothelial dysfunction is present in infants immediately following cardiopulmonary bypass and recovers significantly in some patients within 24 hours postoperatively. Confirmation of an association between persistent endothelial-dependent vasomotor dysfunction and decreased urine output could have important clinical implications. Ongoing research will explore the pattern of endothelial-dependent vasomotor dysfunction after cardiopulmonary bypass and its relationship with biochemical markers of inflammation and clinical outcomes.

Monophosphoryl Lipid a Attenuates Multiorgan Dysfunction During Post-Burn Pseudomonas Aeruginosa Pneumonia in Sheep.

BACKGROUND: Monophosphoryl lipid A (MPLA) is a TLR4 agonist that has potent immunomodulatory properties and modulates innate immune function to improve host resistance to infection with common nosocomial pathogens in mice. The goal of this study was to assess the safety and efficacy of MPLA in a sheep model of burn injury and Pseudomonas aeruginosa pneumonia. The sheep provides a favorable model for preclinical testing as their response to TLR4 agonists closely mimics that of humans. METHODS: Twelve chronically instrumented adult female Merino sheep received 20% total body surface area, third-degree cutaneous burn under anesthesia and analgesia. At 24 h after burn, sheep were randomly allocated to receive: MPLA (2.5 µg/kg i.v., n = 6), or vehicle (i.v., n = 6). At 24 h after MPLA or vehicle treatment, Pseudomonas aeruginosa pneumonia was induced. Sheep were mechanically ventilated, fluid resuscitated and cardiopulmonary variables were monitored for 24 h after induction of pneumonia. Cytokine production, vascular barrier function, and lung bacterial burden were also measured. RESULTS: MPLA infusion induced small and transient alterations in core body temperature, heart rate, pulmonary artery pressure, and pulmonary vascular resistance. Pulmonary mechanics were not altered. Vehicle-treated sheep developed severe acute lung injury during Pseudomonas aeruginosa pneumonia, which was attenuated by MPLA as indicated by improved PaO2/FiO2 ratio, oxygenation index, and shunt fraction. Sheep treated with MPLA also exhibited less vascular leak, lower blood lactate levels, and lower modified organ injury score. MPLA treatment attenuated systemic cytokine production and decreased lung bacterial burden. CONCLUSIONS: MPLA was well tolerated in burned sheep and attenuated development of acute lung injury, lactatemia, cytokinemia, vascular leak, and hemodynamic changes caused by Pseudomonas aeruginosa pneumonia.