Daniel O' Toole

Mesenchymal stromal cells (MSCs) have shown immunomodulatory effects and great promise in many inflammatory diseases such as acute respiratory distress syndrome (ARDS). However, several barriers to translation remain such as cell availability and potency. This study evaluates the therapeutic potentials of three types of MSCs, bone marrow-derived MSCs (BM-MSC), the human induced pluripotent stem cell-derived MSC wild type (iMSC WT) and β2 microglobulin-knockout iMSCs (iMSC B2M KO) with or without proinflammatory cytokine preconditioning. BM-MSC, iMSC WT and iMSC B2M KO were preconditioned with a proinflammatory cytokine cocktail (Cytomix: IL-1β, IFN-γ and TNF-α). Immunoregulatory biomarkers were analysed by flow cytometry and cytokines released by ELISA. MSC antimicrobial properties were analysed via CFU assays while the MSCs’ immunomodulatory effects were evaluated using macrophage activation and T cell proliferation assays. Proinflammatory cytokine preconditioning enhanced the therapeutic potency of all three types of MSCs by increasing immunomodulatory marker expression, enhancing the antimicrobial effects and improving MSC-mediated inhibition of T cell proliferation. These findings provided new insights into the therapeutic potencies of MSCs in inflammation. Further studies are required for in vitro characterisation of the MSCs and in vivo efficacy verification of these MSCs prior to their clinical application.

Dear Frontiers in Medicine reader, Acute respiratory distress syndrome (ARDS) and sepsis remain leading causes of patient morbidity and mortality and the COVID-19 pandemic has highlighted the continuing lack of effective therapeutic options for these and other related acute inflammatory conditions. Among the problems facing ARDS researchers is that there is currently no specific biomarker for rapid diagnosis, and adhering to the Berlin consensus criteria [1] and therefore necessitates methods that are time consuming and expensive, particularly in the context of overloaded health care services. Recent hot topics are subphenotyping of patients, with clearly delineated hyper-and hypo-inflammatory versions of ARDS being more widely recognized [2] and emerging biomarkers of patient outcome giving clinicians the opportunity to treat these quite distinct disease variants with distinct therapeutic approaches. There are also still no licensed medicine specifically targeting ARDS or sepsis [3], a critical gap in the clinician's arsenal and individual organ and symptom support remains the mainstay [4].Recently, a host of novel medicinal approaches have been investigated to address these problems, such as advances in the development of pharmacological agents, recombinant protein drugs and cell and gene therapies. Bioinformatics based approaches and clinical profiling of patients are also paving the way for stratification, targeted therapies and precision medicines. Here, we summarise breaking contributions to the field in a collection of articles published as part of the the Research Topic entitled "Novel Targets and State of the Art Therapies in ARDS and Sepsis".Our first review paper explores the utility of measuring mitochondrial markers of ARDS related disease (McClintock et al, 2022). The summarized studies include assessments of mitochondrial DNA in blood, peroxidation markers and a range of metabolites such as glucose, lactate and xanthine and point to a future where simple point of care devices could instantly diagnose ARDS and ARDS severity based on minimal essential parameters. In a patient sample analysis study, Peng and colleagues have identified dysfunctional iron metabolism mediated via hepcidin as a predictor of patient outcome in COVID-19 ARDS (Peng et al, 2022), a finding which could ultimately be applicable to ARDS of any aetiology. Finally in this group of manuscripts we have a study of immune cell subpopulations in ARDS patients where it was discovered that the ratio of CD4/CD8 markers was an effective predictor of disease severity and could assist in directing resources and appropriate care to specific sufferers (Pascual-Dapena et al, 2022).Our second thematic grouping of papers is a deep dive into the pathology and pathobiology of ARDS. Indeed, it could be argued that this topic overlaps with and informs diagnostics and therapeutics and is fundamental to an intelligent approach to ARDS patient care. As well as the alveolar cells of the lung, acute lung injury is also associated with endothelial dysfunction and vascular thrombosis and we are provided with a comprehensive overview of how the Kallikrein-Kinin axis contributes to this disease process (Bailey et al, 2023). Large datasets demand increasingly complex computational approaches to maximize the meaningful information extracted, and so we are happy to welcome from Parkinson and colleagues a machine learning assisted mRNA profiling of one of the more vulnerable patient populations, that of neonatal sepsis (Parkinson et al, 2023). We also see a single-cell analysis approach to assessment of risk factors for progression of shock to ARDS that has identified the importance of chromatin accessibility near a specific gene locus, CALCRL (Armstead et al, 2023), To round off this section, we have two studies focusing on specific disease mechanisms and their involvement in ARDS and sepsis. Firstly, Liu et al. elucidates the contribution of the ferroptosis pathway in ischemia/reperfusion driven inflammation in a rat model (Liu et al, 2023) and secondly we have from Hu and coworkers an intriguing paper detailing the involvement of the C-type lectin pancreatic stone protein in multiple organ dysfunction syndrome (MODS) (Hu et al, 2023).In our final subsection we explore approaches to ARDS and sepsis patient management and therapeutics, from refining traditional support protocols to cutting edge advanced therapeutic medicinal products (ATMPs). This theme includes a retrospective sepsis patient analysis comparing saline and Ringers solutions for resuscitation (Isha et al, 2023), followed with a preclinical study from Gonzalez and colleagues of a nebulizer delivered stem cell therapy for ARDS (González et al, 2023), this with the novelty of utilizing the secretome as opposed to the cell itself.We, the editors of this special edition of Frontiers in Medicine, hope that you, the reader, find this collection to be as informative and interesting as we did when assembling and curating it, and expect that it will spark future research into diagnosing and treating this devastating family of diseases.Dr. Daniel O'Toole. Dr. Shahd Horie. Dr. Emma Murphy.

Background: Despite promise in preclinical models of acute respiratory distress syndrome (ARDS), mesenchymal stem cells (MSC) have failed to translate to therapeutic benefit in clinical trials. The MSC is a live cell medicine and interacts with the patient’s disease state. Here, we explored this interaction, seeking to devise strategies to enhance MSC therapeutic function. Methods: Human bone-marrow-derived MSCs were exposed to lung homogenate from healthy and E. coli-induced ARDS rat models. Apoptosis and functional assays of the MSCs were performed. Results: The ARDS model showed reduced arterial oxygenation, decreased lung compliance and an inflammatory microenvironment compared to controls. MSCs underwent more apoptosis after stimulation by lung homogenate from controls compared to E. coli, which may explain why MSCs persist longer in ARDS subjects after administration. Changes in expression of cell surface markers and cytokines were associated with lung homogenate from different groups. The anti-microbial effects of MSCs did not change with the stimulation. Moreover, the conditioned media from lung-homogenate-stimulated MSCs inhibited T-cell proliferation. Conclusions: These findings suggest that the ARDS microenvironment plays an important role in the MSC’s therapeutic mechanism of action, and changes can inform strategies to modulate MSC-based cell therapy for ARDS.

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.

Background: Mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) have been proposed as an alternative to cell therapy, creating new possible delivery modalities such as nebulisation. We wished to investigate the therapeutic potential of directly nebulised MSC-EVs in the mitigation of Escherichiacoli-induced pneumonia. Methods: EV size, surface markers and miRNA content were assessed pre- and post-nebulisation. BEAS2B and A459 lung cells were exposed to lipopolysaccharide (LPS) and treated with nebulised bone marrow (BM) or umbilical cord (UC) MSC-EVs. Viability assays (MTT) and inflammatory cytokine assays were performed. THP-1 monocytes were stimulated with LPS and nebulised BM- or UC-EVs and phagocytosis activity was measured. For in vivo experiments, mice received LPS intratracheally (IT) followed by BM- or UC-EVs intravenously (IV) and injury markers assessed at 24 h. Rats were instilled with E. coli bacteria IT and BM- or UC-EVs delivered IV or by direct nebulisation. At 48 h, lung damage was assessed by physiological parameters, histology and inflammatory marker presence. Results: MSC-EVs retained their immunomodulatory and wound healing capacity after nebulisation in vitro. EV integrity and content were also preserved. Therapy with IV or nebulised MSC-EVs reduced the severity of LPS-induced lung injury and E. coli-induced pneumonia by reducing bacterial load and oedema, increasing blood oxygenation and improving lung histological scores. MSC-EV treated animals also showed lower levels of inflammatory cytokines and inflammatory-related markers. Conclusions: MSC-EVs given IV attenuated LPS-induced lung injury, and nebulisation of MSC-EVs did not affect their capacity to attenuate lung injury caused by E. coli pneumonia, as evidenced by reduction in bacterial load and improved lung physiology.

Background: Mesenchymal stem cells (MSC) have shown immense therapeutic promise in a range of inflammatory diseases, including acute respiratory distress syndrome (ARDS), and are rapidly advancing through clinical trials. Among their multimodal mechanisms of action, MSCs exert strong immunomodulatory effects via their secretome, which contains cytokines, small molecules, extracellular vesicles, and a range of other factors. Recent studies have shown that the MSC secretome can recapitulate many of the beneficial effects of the MSC itself. We aimed to determine the therapeutic capacity of the MSC secretome in a rat bacterial pneumonia model, especially when delivered directly to the lung by nebulization which is a technique more appropriate for the ventilated patient.Methods: Conditioned medium (CM) was generated from human bone marrow derived MSCs in the absence of antibiotics and serum supplements. Post-nebulization lung penetration was estimated through nebulization of CM to a cascade impactor and simulated lung and quantification of collected total protein and IL-8 cytokine. Control and nebulized CM was added to a variety of lung cell culture models and injury resolution assessed. In a rat E. coli pneumonia model, CM was instilled or administered by nebulization and lung injury and inflammation assessed at 48 h.Results: MSC-CM was predicted to have good distal lung penetration and delivery when administered by nebulizer. Both control and nebulized CM reduced NF-κB activation and inflammatory cytokine production in lung cell culture, while promoting cell viability and would closure in oxidative stress and scratch wound models. In a rat bacterial pneumonia model, both instilled and nebulizer delivered CM improved lung function, increasing blood oxygenation and reducing carbon dioxide levels compared to unconditioned medium controls. A reduction in bacterial load was also observed in both treatment groups. Inflammatory cytokines were reduced significantly by both liquid and aerosol CM administration, with less IL-1β, IL-6, and CINC1 in these groups compared to controls.Conclusion: MSC-CM is a potential therapeutic for pneumonia ARDS, and administration is compatible with vibrating mesh nebulization.

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.

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.