Aerosol Delivery of a Novel Recombinant Modified Superoxide Dismutase Protein Reduces Oxidant Injury and Attenuates Escherichia coli Induced Lung Injury in Rats.

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory failure syndrome with diverse etiologies characterized by increased permeability of alveolar-capillary membranes, pulmonary edema, and acute onset hypoxemia. During the ARDS acute phase, neutrophil infiltration into the alveolar space results in uncontrolled release of reactive oxygen species (ROS) and proteases, overwhelming antioxidant defenses and causing alveolar epithelial and lung endothelial injury. Objectives: To investigate the therapeutic potential of a novel recombinant human Cu-Zn-superoxide dismutase (SOD) fusion protein in protecting against ROS injury and for aerosolized SOD delivery to treat Escherichia coli induced ARDS. Methods: Fusion proteins incorporating human Cu-Zn-SOD (hSOD1), with (pep1-hSOD1-his) and without (hSOD1-his) a fused hyaluronic acid-binding peptide, were expressed in E. coli. Purified proteins were evaluated in in vitro assays with human bronchial epithelial cells and through aerosolized delivery to the lung of an E. coli-induced ARDS rat model. Results: SOD proteins exhibited high SOD activity in vitro and protected bronchial epithelial cells from oxidative damage. hSOD1-his and pep1-hSOD1-his retained SOD activity postnebulization and exhibited no adverse effects in the rat. Pep1-hSOD1-his administered through instillation or nebulization to the lung of an E. coli-induced pneumonia rat improved arterial oxygenation and lactate levels compared to vehicle after 48 hours. Static lung compliance was improved when the pep1-hSOD1-his protein was delivered by instillation. White cell infiltration to the lung was significantly reduced by aerosolized delivery of protein, and reduction of cytokine-induced neutrophil chemoattractant-1, interferon-gamma, and interleukin 6 pro-inflammatory cytokine concentrations in bronchoalveolar lavage was observed. Conclusions: Aerosol delivery of a novel recombinant modified SOD protein reduces oxidant injury and attenuates E. coli induced lung injury in rats. The results provide a strong basis for further investigation of the therapeutic potential of hSOD1 in the treatment of ARDS.

Differential Effects of Cytokine Versus Hypoxic Preconditioning of Human Mesenchymal Stromal Cells in Pulmonary Sepsis Induced by Antimicrobial-Resistant Klebsiella pneumoniae.

Background: Pulmonary sepsis is a leading cause of hospital mortality, and sepses arising from antimicrobial-resistant (AMR) bacterial strains are particularly difficult to treat. Here we investigated the potential of mesenchymal stromal cells (MSCs) to combat established Klebsiella pneumoniae pneumosepsis and further evaluated MSC preconditioning and pre-activation methods. Methods: The potential for naïve and preconditioned MSCs to enhance wound healing, reduce inflammation, preserve metabolic activity, and enhance bacterial killing was assessed in vitro. Rats were subjected to intratracheal K. pneumoniae followed by the intravenous administration of MSCs. Physiological indices, blood, bronchoalveolar lavage (BAL), and tissues were obtained 72 h later. Results: In vitro assays confirmed that preconditioning enhances MSC function, accelerating pulmonary epithelial wound closure, reducing inflammation, attenuating cell death, and increasing bacterial killing. Cytomix-pre-activated MSCs are superior to naïve and hypoxia-exposed MSCs in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, reducing bacteria, and attenuating histologic injuries in lungs. BAL inflammatory cytokines were reduced, correlating with decreases in polymorphonuclear (PMN) cells. MSCs increased PMN apoptosis and the CD4:CD8 ratio in BAL. Systemically, granulocytes, classical monocytes, and the CD4:CD8 ratio were reduced, and nonclassical monocytes were increased. Conclusions: Preconditioning with cytokines, but not hypoxia, enhances the therapeutic potential of MSCs in clinically relevant models of K. pneumoniae-induced pneumosepsis.

Multiple Dosing and Preactivation of Mesenchymal Stromal Cells Enhance Efficacy in Established Pneumonia Induced by Antimicrobial-Resistant Klebsiella pneumoniae in Rodents.

Antimicrobial-resistant (AMR) bacteria, such as Klebsiella species, are an increasingly common cause of hospital-acquired pneumonia, resulting in high mortality and morbidity. Harnessing the host immune response to AMR bacterial infection using mesenchymal stem cells (MSCs) is a promising approach to bypass bacterial AMR mechanisms. The administration of single doses of naïve MSCs to ARDS clinical trial patient cohorts has been shown to be safe, although efficacy is unclear. The study tested whether repeated MSC dosing and/or preactivation, would attenuate AMR Klebsiella pneumonia-induced established pneumonia. Rat models of established K. pneumoniae-induced pneumonia were randomised to receive intravenous naïve or cytomix-preactivated umbilical cord MSCs as a single dose at 24 h post pneumonia induction with or without a subsequent dose at 48 h. Physiological indices, bronchoalveolar lavage (BAL), and tissues were obtained at 72 h post pneumonia induction. A single dose of naïve MSCs was largely ineffective, whereas two doses of MSCs were effective in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, while reducing bacteria and injury in the lung. Cytomix-preactivated MSCs were superior to naïve MSCs. BAL neutrophil counts and activation were reduced, and apoptosis increased. MSC therapy reduced cytotoxic BAL T cells, and increased CD4+/CD8+ ratios. Systemically, granulocytes, classical monocytes, and the CD4+/CD8+ ratio were reduced, and nonclassical monocytes were increased. Repeated doses of MSCs-particularly preactivated MSCs-enhance their therapeutic potential in a clinically relevant model of established AMR K. pneumoniae-induced pneumosepsis.

Aerosolized Pulmonary Delivery of mRNA Constructs Attenuates Severity of Escherichia coli Pneumonia in the Rat.

Acute respiratory distress syndrome (ARDS), a rapid onset inflammatory lung disease with no effective specific therapy, typically has pathogenic etiology termed pneumonia. In previous studies nuclear factor-κB (NF-κB) inhibitor α super-repressor (IκBα-SR) and extracellular superoxide dismutase 3 (SOD3) reduced pneumonia severity when prophylactically delivered by viral vector. In this study, mRNA coding for green fluorescent protein, IκBα-SR, or SOD3 was complexed with cationic lipid, passed through a vibrating mesh nebulizer, and delivered to cell culture or directly to rats undergoing Escherichia coli pneumonia. Injury level was then assessed at 48 h. In vitro, expression was observed as early as 4 h in lung epithelial cells. IκBα-SR and wild-type IκBα mRNAs attenuated inflammatory markers, while SOD3 mRNA induced protective and antioxidant effects. In rat E. coli pneumonia, IκBα-SR mRNA reduced arterial carbon dioxide (pCO2) and reduced lung wet/dry ratio. SOD3 mRNA improved static lung compliance and alveolar-arterial oxygen gradient (AaDO2) and decreased bronchoalveolar lavage (BAL) bacteria load. White cell infiltration and inflammatory cytokine concentrations in BAL and serum were reduced by both mRNA treatments compared to scrambled mRNA controls. These findings indicate nebulized mRNA therapeutics are a promising approach to ARDS therapy, with rapid expression of protein and observable amelioration of pneumonia symptoms.

Intra-vital imaging of mesenchymal stromal cell kinetics in the pulmonary vasculature during infection.

Mesenchymal stem/stromal cells (MSCs) have demonstrated efficacy in pre-clinical models of inflammation and tissue injury, including in models of lung injury and infection. Rolling, adhesion and transmigration of MSCs appears to play a role during MSC kinetics in the systemic vasculature. However, a large proportion of MSCs become entrapped within the lungs after intravenous administration, while the initial kinetics and the site of arrest of MSCs in the pulmonary vasculature are unknown. We examined the kinetics of intravascularly administered MSCs in the pulmonary vasculature using a microfluidic system in vitro and intra-vital microscopy of intact mouse lung. In vitro, MSCs bound to endothelium under static conditions but not under laminar flow. VCAM-1 antibodies did not affect MSC binding. Intravital microscopy demonstrated MSC arrest at pulmonary micro-vessel bifurcations due to size obstruction. Retention of MSCs in the pulmonary microvasculature was increased in Escherichia coli-infected animals. Trapped MSCs deformed over time and appeared to release microvesicles. Labelled MSCs retained therapeutic efficacy against pneumonia. Our results suggest that MSCs are physically obstructed in pulmonary vasculature and do not display properties of rolling/adhesion, while retention of MSCs in the infected lung may require receptor interaction.

Mesenchymal Stem/Stromal Cells Therapy for Sepsis and Acute Respiratory Distress Syndrome.

Sepsis and acute respiratory distress syndrome (ARDS) constitute devastating conditions with high morbidity and mortality. Sepsis results from abnormal host immune response, with evidence for both pro- and anti-inflammatory activation present from the earliest phases. The "proinflammatory" response predominates initially causing host injury, with later-phase sepsis characterized by immune cell hypofunction and opportunistic superinfection. ARDS is characterized by inflammation and disruption of the alveolar-capillary membrane leading to injury and lung dysfunction. Sepsis is the most common cause of ARDS. Approximately 20% of deaths worldwide in 2017 were due to sepsis, while ARDS occurs in over 10% of all intensive care unit patients and results in a mortality of 30 to 45%. Given the fact that sepsis and ARDS share some-but not all-underlying pathophysiologic injury mechanisms, the lack of specific therapies, and their frequent coexistence in the critically ill, it makes sense to consider therapies for both conditions together. In this article, we will focus on the therapeutic potential of mesenchymal stem/stromal cells (MSCs). MSCs are available from several tissues, including bone marrow, umbilical cord, and adipose tissue. Allogeneic administration is feasible, an important advantage for acute conditions like sepsis or ARDS. They possess diverse mechanisms of action of relevance to sepsis and ARDS, including direct and indirect antibacterial actions, potent effects on the innate and adaptive response, and pro-reparative effects. MSCs can be preactivated thereby potentiating their effects, while the use of their extracellular vesicles can avoid whole cell administration. While early-phase clinical trials suggest safety, considerable challenges exist in moving forward to phase III efficacy studies, and to implementation as a therapy should they prove effective.

Umbilical Cord-Derived CD362+ Mesenchymal Stromal Cells Attenuate Polymicrobial Sepsis Induced by Caecal Ligation and Puncture.

Mesenchymal stromal cells (MSCs) have a multimodal, immunomodulatory mechanism of action and are now in clinical trials for single organ and systemic sepsis. However, a number of practicalities around source, homogeneity and therapeutic window remain to be determined. Here, we utilised conditioned medium from CD362+-sorted umbilical cord-human MSCs (UC-hMSCs) for a series of in vitro anti-inflammatory assays and the cryopreserved MSCs themselves in a severe (Series 1) or moderate (Series 2+3) caecal ligation and puncture (CLP) rodent model. Surviving animals were assessed at 48 h post injury induction. MSCs improved human lung, colonic and kidney epithelial cell survival following cytokine activation. In severe systemic sepsis, MSCs administered at 30 min enhanced survival (Series 1), and reduced organ bacterial load. In moderate systemic sepsis (Series 2), MSCs were ineffective when delivered immediately or 24 h later. Of importance, MSCs delivered 4 h post induction of moderate sepsis (Series 3) were effective, improving serum lactate, enhancing bacterial clearance from tissues, reducing pro-inflammatory cytokine concentrations and increasing antimicrobial peptides in serum. While demonstrating benefit and immunomodulation in systemic sepsis, therapeutic efficacy may be limited to a specific point of disease onset, and repeat dosing, MSC enhancement or other contingencies may be necessary.

The role of cells and their products in respiratory drug delivery: the past, present, and future.

INTRODUCTION: Cell-based delivery systems offer considerable promise as novel and innovative therapeutics to target the respiratory system. These systems consist of cells and/or their extracellular vesicles that deliver their contents, such as anti-microbial peptides, micro RNAs, and even mitochondria to the lung, exerting direct therapeutic effects. AREAS COVERED: The purpose of this article is to critically review the status of cell-based therapies in the delivery of therapeutics to the lung, evaluate current progress, and elucidate key challenges to the further development of these novel approaches. An overview as to how these cells and/or their products may be modified to enhance efficacy is given. More complex delivery cell-based systems, including cells or vesicles that are genetically modified to (over)express specific therapeutic products, such as proteins and therapeutic nucleic acids are also discussed. Focus is given to the use of the aerosol route to deliver these products directly into the lung. EXPERT OPINION: The use of biological carriers to deliver chemical or biological agents demonstrates great potential in modern medicine. The next generation of drug delivery systems may comprise 'cell-inspired' drug carriers that are entirely synthetic, developed using insights from cell-based therapeutics to overcome limitations of current generation synthetic carriers.

Demographics, management and outcome of females and males with acute respiratory distress syndrome in the LUNG SAFE prospective cohort study.

RATIONALE: We wished to determine the influence of sex on the management and outcomes in acute respiratory distress syndrome (ARDS) patients in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE). METHODS: We assessed the effect of sex on mortality, intensive care unit and hospital length of stay, and duration of invasive mechanical ventilation (IMV) in patients with ARDS who underwent IMV, adjusting for plausible clinical and geographic confounders. FINDINGS: Of 2377 patients with ARDS, 905 (38%) were female and 1472 (62%) were male. There were no sex differences in clinician recognition of ARDS or critical illness severity profile. Females received higher tidal volumes (8.2±2.1 versus 7.2±1.6 mL·kg-1; p<0.0001) and higher plateau and driving pressures compared with males. Lower tidal volume ventilation was received by 50% of females compared with 74% of males (p<0.0001). In shorter patients (height ≤1.69 m), females were significantly less likely to receive lower tidal volumes. Surviving females had a shorter duration of IMV and reduced length of stay compared with males. Overall hospital mortality was similar in females (40.2%) versus males (40.2%). However, female sex was associated with higher mortality in patients with severe confirmed ARDS (OR for sex (male versus female) 0.35, 95% CI 0.14-0.83). CONCLUSIONS: Shorter females with ARDS are less likely to receive lower tidal volume ventilation, while females with severe confirmed ARDS have a higher mortality risk. These data highlight the need for better ventilatory management in females to improve their outcomes from ARDS.

Modulating the distribution and fate of exogenously delivered MSCs to enhance therapeutic potential: knowns and unknowns.

Mesenchymal stem/stromal cells (MSCs) are undergoing intensive translational research for several debilitating conditions, including critical illnesses such as ARDS and sepsis. MSCs exert diverse biologic effects via their interaction with host tissues, via mechanisms that require the MSC to be in close proximity to the area of injury. Fully harnessing the therapeutic potential of advanced medicinal therapeutic products such as MSCs and their successful translation to clinical use requires a detailed understanding of MSC distribution and persistence in the injured tissues.Key aspects include understanding MSC distribution within the body, the response of the host to MSC administration, and the ultimate fate of exogenously administered MSCs within the host. Factors affecting this interaction include the MSC tissue source, the in vitro MSC culture conditions, the route of MSC administration and the specific issues relating to the target disease state, each of which remains to be fully characterised. Understanding these factors may generate strategies to modify MSC distribution and fate that may enhance their therapeutic effect.This review will examine our understanding of the mechanisms of action of MSCs, the early and late phase distribution kinetics of MSCs following in vivo administration, the ultimate fate of MSCs following administration and the potential importance of these MSC properties to their therapeutic effects. We will critique current cellular imaging and tracking methodologies used to track exogenous MSCs and their suitability for use in patients, discuss the insights they provide into the distribution and fate of MSCs after administration, and suggest strategies by which MSC biodistribution and fate may be modulated for therapeutic effect and clinical use.In conclusion, a better understanding of patterns of biodistribution and of the fate of MSCs will add important additional safety data regarding MSCs, address regulatory requirements, and may uncover strategies to increase the distribution and/or persistence of MSC at the sites of injury, potentially increasing their therapeutic potential for multiple disorders.

Cell therapy in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is driven by a severe pro-inflammatory response resulting in lung damage, impaired gas exchange and severe respiratory failure. There is no specific treatment that effectively improves outcome in ARDS. However, in recent years, cell therapy has shown great promise in preclinical ARDS studies. A wide range of cells have been identified as potential candidates for use, among these are mesenchymal stromal cells (MSCs), which are adult multi-lineage cells that can modulate the immune response and enhance repair of damaged tissue. The therapeutic potential of MSC therapy for sepsis and ARDS has been demonstrated in multiple in vivo models. The therapeutic effect of these cells seems to be due to two different mechanisms; direct cellular interaction, and paracrine release of different soluble products such as extracellular vesicles (EVs)/exosomes. Different approaches have also been studied to enhance the therapeutic effect of these cells, such as the over-expression of anti-inflammatory or pro-reparative molecules. Several clinical trials (phase I and II) have already shown safety of MSCs in ARDS and other diseases. However, several translational issues still need to be addressed, such as the large-scale production of cells, and their potentiality and variability, before the therapeutic potential of stem cells therapies can be realized.