Mesenchymal stromal cell extracellular vesicles improve lung development in mechanically ventilated preterm lambs.

Novel therapies are needed for bronchopulmonary dysplasia (BPD) because no effective treatment exists. Mesenchymal stromal cell extracellular vesicles (MSC-sEVs) have therapeutic efficacy in a mouse pup neonatal hyperoxia BPD model. We tested the hypothesis that MSC-sEVs will improve lung functional and structural development in mechanically ventilated preterm lambs. Preterm lambs (∼129 days; equivalent to human lung development at ∼28 wk gestation) were exposed to antenatal steroids, surfactant, caffeine, and supported by mechanical ventilation for 6-7 days. Lambs were randomized to blinded treatment with either MSC-sEVs (human bone marrow MSC-derived; 2 × 1011 particles iv; n = 8; 4 F/4 M) or vehicle control (saline iv; 4 F/4 M) at 6 and 78 h post delivery. Physiological targets were pulse oximetry O2 saturation 90-94% ([Formula: see text] 60-90 mmHg), [Formula: see text] 45-60 mmHg (pH 7.25-7.35), and tidal volume 5-7 mL/kg. MSC-sEVs-treated preterm lambs tolerated enteral feedings compared with vehicle control preterm lambs. Differences in weight patterns were statistically significant. Respiratory severity score, oxygenation index, A-a gradient, distal airspace wall thickness, and smooth muscle thickness around terminal bronchioles and pulmonary arterioles were significantly lower for the MSC-sEVs group. S/F ratio, radial alveolar count, secondary septal volume density, alveolar capillary surface density, and protein abundance of VEGF-R2 were significantly higher for the MSC-sEVs group. MSC-sEVs improved respiratory system physiology and alveolar formation in mechanically ventilated preterm lambs. MSC-sEVs may be an effective and safe therapy for appropriate functional and structural development of the lung in preterm infants who require mechanical ventilation and are at risk of developing BPD.NEW & NOTEWORTHY This study focused on potential treatment of preterm infants at risk of developing bronchopulmonary dysplasia (BPD), for which no effective treatment exists. We tested treatment of mechanically ventilated preterm lambs with human mesenchymal stromal cell extracellular vesicles (MSC-sEVs). The results show improved respiratory gas exchange and parenchymal growth of capillaries and epithelium that are necessary for alveolar formation. Our study provides new mechanistic insight into potential efficacy of MSC-sEVs for preterm infants at risk of developing BPD.

Photoreceptors inhibit pathological retinal angiogenesis through transcriptional regulation of Adam17 via c-Fos.

Pathological retinal angiogenesis profoundly impacts visual function in vascular eye diseases, such as retinopathy of prematurity (ROP) in preterm infants and age-related macular degeneration in the elderly. While the involvement of photoreceptors in these diseases is recognized, the underlying mechanisms remain unclear. This study delved into the pivotal role of photoreceptors in regulating abnormal retinal blood vessel growth using an oxygen-induced retinopathy (OIR) mouse model through the c-Fos/A disintegrin and metalloprotease 17 (Adam17) axis. Our findings revealed a significant induction of c-Fos expression in rod photoreceptors, and c-Fos depletion in these cells inhibited pathological neovascularization and reduced blood vessel leakage in the OIR mouse model. Mechanistically, c-Fos directly regulated the transcription of Adam17 a shedding protease responsible for the production of bioactive molecules involved in inflammation, angiogenesis, and cell adhesion and migration. Furthermore, we demonstrated the therapeutic potential by using an adeno-associated virus carrying a rod photoreceptor-specific short hairpin RNA against c-fos which effectively mitigated abnormal retinal blood vessel overgrowth, restored retinal thickness, and improved electroretinographic (ERG) responses. In conclusion, this study highlights the significance of photoreceptor c-Fos in ROP pathology, offering a novel perspective for the treatment of this disease.

SOCS3 regulates pathological retinal angiogenesis through modulating SPP1 expression in microglia and macrophages.

Pathological ocular angiogenesis has long been associated with myeloid cell activation. However, the precise cellular and molecular mechanisms governing the intricate crosstalk between the immune system and vascular changes during ocular neovascularization formation remain elusive. In this study, we demonstrated that the absence of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells led to a substantial accumulation of microglia and macrophage subsets during the neovascularization process. Our single-cell RNA sequencing data analysis revealed a remarkable increase in the expression of the secreted phosphoprotein 1 (Spp1) gene within these microglia and macrophages, identifying subsets of Spp1-expressing microglia and macrophages during neovascularization formation in angiogenesis mouse models. Notably, the number of Spp1-expressing microglia and macrophages exhibited further elevation during neovascularization in mice lacking myeloid SOCS3. Moreover, our investigation unveiled the Spp1 gene as a direct transcriptional target gene of signal transducer and activator of transcription 3. Importantly, pharmaceutical activation of SOCS3 or blocking of SPP1 resulted in a significant reduction in pathological neovascularization. In conclusion, our study highlights the pivotal role of the SOCS3/STAT3/SPP1 axis in the regulation of pathological retinal angiogenesis.

Immunoregulatory macrophages modify local pulmonary immunity and ameliorate hypoxic-pulmonary hypertension.

Rationale: Macrophages play a central role in the onset and progression of vascular disease in pulmonary hypertension (PH) and cell-based immunotherapies aimed at treating vascular remodeling are lacking. Objective: To evaluate the effect of pulmonary administration of macrophages modified to have an anti-inflammatory/pro-resolving phenotype in attenuating early pulmonary inflammation and progression of experimentally induced PH. Methods: Mouse bone marrow derived macrophages (BMDMs) were polarized in vitro to a regulatory (M2 reg ) phenotype. M2 reg profile and anti-inflammatory capacity were assessed in vitro upon lipopolysaccharide (LPS)/interferon-γ (IFNγ) restimulation, before their administration to 8- to 12-week-old mice. M2 reg protective effect was tested at early (2 to 4 days) and late (4 weeks) time points during hypoxia (8.5% O 2 ) exposure. Levels of inflammatory markers were quantified in alveolar macrophages and whole lung, while PH development was ascertained by right ventricular systolic pressure (RSVP) and right ventricular hypertrophy (RVH) measurements. Bronchoalveolar lavage (BAL) from M2 reg -transplanted hypoxic mice was collected, and its inflammatory potential tested on naïve BMDMs. Results: M2 reg macrophages demonstrated a stable anti-inflammatory phenotype upon a subsequent pro-inflammatory stimulus by maintaining the expression of specific anti-inflammatory markers (Tgfß, Il10 and Cd206) and downregulating the induction of proinflammatory cytokines and surface molecules (Cd86, Il6 and Tnfα). A single dose of M2 regs attenuated the hypoxic monocytic recruitment and perivascular inflammation. Early hypoxic lung and alveolar macrophage inflammation leading to PH development was significantly reduced and, importantly, M2 regs attenuated RVH, RVSP and vascular remodeling at 4 weeks post treatment. Conclusions: Adoptive transfer of M2 regs halts the recruitment of monocytes and modifies the hypoxic lung microenvironment, potentially changing the immunoreactivity of recruited macrophages and restoring normal immune functionality of the lung. These findings provide new mechanistic insights on the diverse role of macrophage phenotype on lung vascular homeostasis that can be explored as novel therapeutic targets.

Harnessing the therapeutic potential of the stem cell secretome in neonatal diseases.

Preterm birth and intrapartum related complications account for a substantial amount of mortality and morbidity in the neonatal period despite significant advancements in neonatal-perinatal care. Currently, there is a noticeable lack of curative or preventative therapies available for any of the most common complications of prematurity including bronchopulmonary dysplasia, necrotizing enterocolitis, intraventricular hemorrhage, periventricular leukomalacia and retinopathy of prematurity or hypoxic-ischemic encephalopathy, the main cause of perinatal brain injury in term infants. Mesenchymal stem/stromal cell-derived therapy has been an active area of investigation for the past decade and has demonstrated encouraging results in multiple experimental models of neonatal disease. It is now widely acknowledged that mesenchymal stem/stromal cells exert their therapeutic effects via their secretome, with the principal vector identified as extracellular vesicles. This review will focus on summarizing the current literature and investigations on mesenchymal stem/stromal cell-derived extracellular vesicles as a treatment for neonatal diseases and examine the considerations to their application in the clinical setting.

Mesenchymal stromal cell-derived syndecan-2 regulates the immune response during sepsis to foster bacterial clearance and resolution of inflammation.

Sepsis is a life-threatening process related to a dysregulated host response to an underlying infection, which results in organ dysfunction and poor outcomes. Therapeutic strategies using mesenchymal stromal cells (MSCs) are under investigation for sepsis, with efforts to improve cellular utility. Syndecan (SDC) proteins are transmembrane proteoglycans involved with cellular signaling events including tissue repair and modulating inflammation. Bone marrow-derived human MSCs express syndecan-2 (SDC2) at a level higher than other SDC family members; thus, we explored SDC2 in MSC function. Administration of human MSCs silenced for SDC2 in experimental sepsis resulted in decreased bacterial clearance, and increased tissue injury and mortality compared with wild-type MSCs. These findings were associated with a loss of resolution of inflammation in the peritoneal cavity, and higher levels of proinflammatory mediators in organs. MSCs silenced for SDC2 had a decreased ability to promote phagocytosis of apoptotic neutrophils by macrophages in the peritoneum, and also a diminished capability to convert macrophages from a proinflammatory to a proresolution phenotype via cellular or paracrine actions. Extracellular vesicles are a paracrine effector of MSCs that may contribute to resolution of inflammation, and their production was dramatically reduced in SDC2-silenced human MSCs. Collectively, these data demonstrate the importance of SDC2 for cellular and paracrine function of human MSCs during sepsis.

Antenatal Mesenchymal Stromal Cell Extracellular Vesicle Therapy Prevents Preeclamptic Lung Injury in Mice.

In preeclamptic pregnancies, a variety of intrauterine alterations lead to abnormal placentation, release of inflammatory and/or antiangiogenic factors, and subsequent fetal growth restriction with significant potential to cause a primary insult to the developing fetal lung. Thus, modulation of the maternal intrauterine environment may be a key therapeutic avenue to prevent preeclampsia-associated developmental lung injury. A biologic therapy of interest is mesenchymal stromal cell-derived extracellular vesicles (MEx), which we have previously shown to ameliorate preeclamptic physiology through intrauterine immunomodulation. To evaluate the therapeutic potential of MEx to improve developmental lung injury in experimental preeclampsia, using the heme oxygenase-1-null mouse (Hmox1-/-) model, preeclamptic pregnant dams were administered intravenous antenatal MEx treatment during each week of pregnancy followed by analysis of fetal and postnatal lung tissues, amniotic fluid protein profiles, and lung explant and amniotic fluid cocultures in comparison with control and untreated preeclamptic pregnancies. We first identified that a preeclamptic intrauterine environment had a significant adverse impact on fetal lung development, including alterations in fetal lung developmental gene profiles in addition to postnatal alveolar and bronchial changes. Amniotic fluid proteomic analysis and fetal lung explant and amniotic fluid cocultures further demonstrated that maternally administered MEx altered the expression of multiple inflammatory mediators in the preeclamptic intrauterine compartment, resulting in the normalization of fetal lung branching morphogenesis and developmental gene expression. Our evaluation of fetal and postnatal parameters overall suggests that antenatal MEx treatment may provide a highly valuable preventative therapeutic modality for amelioration of lung development in preeclamptic disease.

Antenatal mesenchymal stromal cell extracellular vesicle treatment preserves lung development in a model of bronchopulmonary dysplasia due to chorioamnionitis.

Antenatal stressors such as chorioamnionitis (CA) increase the risk for bronchopulmonary dysplasia (BPD). Studies have shown that experimental BPD can be ameliorated by postnatal treatment with mesenchymal stromal cell-derived extracellular vesicles (MEx). However, the antenatal efficacy of MEx to prevent BPD is unknown. To determine whether antenatal MEx therapy attenuates intrauterine inflammation and preserves lung growth in a rat model of CA-induced BPD. At embryonic day (E)20, rat litters were treated with intra-amniotic injections of saline, endotoxin (ETX) to model chorioamnionitis, MEx, or ETX plus MEx followed by cesarean section delivery with placental harvest at E22. Placental and lung evaluations were conducted at day 0 and day 14, respectively. To assess the effects of ETX and MEx on lung growth in vitro, E15 lung explants were imaged for distal branching. Placental tissues from ETX-exposed pregnancies showed increased expression of inflammatory markers NLRP-3 and IL-1ß and altered spiral artery morphology. In addition, infant rats exposed to intrauterine ETX had reduced alveolarization and pulmonary vessel density (PVD), increased right ventricular hypertrophy (RVH), and decreased lung mechanics. Intrauterine MEx therapy of ETX-exposed pups reduced inflammatory cytokines, normalized spiral artery architecture, and preserved distal lung growth and mechanics. In vitro studies showed that MEx treatment enhanced distal lung branching and increased VEGF and SPC gene expression. Antenatal MEx treatment preserved distal lung growth and reduced intrauterine inflammation in a model of CA-induced BPD. We speculate that MEx may provide a novel therapeutic strategy to prevent BPD due to antenatal inflammation.

Extracellular Vesicles Protect the Neonatal Lung from Hyperoxic Injury through the Epigenetic and Transcriptomic Reprogramming of Myeloid Cells.

Rationale: Mesenchymal stem/stromal cell (MSC)-small extracellular vesicle (MEx) treatment has shown promise in experimental models of neonatal lung injury. The molecular mechanisms by which MEx afford beneficial effects remain incompletely understood. Objectives: To investigate the therapeutic mechanism of action through assessment of MEx biodistribution and impact on immune cell phenotypic heterogeneity. Methods: MEx were isolated from the conditioned medium of human umbilical cord Wharton's jelly-derived MSCs. Newborn mice were exposed to hyperoxia (HYRX, 75% O2) from birth and returned to room air at Postnatal Day 14 (PN14). Mice received either a bolus intravenous MEx dose at PN4 or bone marrow-derived myeloid cells (BMDMy) pretreated with MEx. Animals were killed at PN4, PN7, PN14, or PN28 to characterize MEx biodistribution or for assessment of pulmonary parameters. The therapeutic role of MEx-educated BMDMy was determined in vitro and in vivo. Measurements and Main Results: MEx therapy ameliorated core histological features of HYRX-induced neonatal lung injury. Biodistribution and mass cytometry studies demonstrated that MEx localize in the lung and interact with myeloid cells. MEx restored the apportion of alveolar macrophages in the HYRX-injured lung and concomitantly suppressed inflammatory cytokine production. In vitro and ex vivo studies revealed that MEx promoted an immunosuppressive BMDMy phenotype. Functional assays demonstrated that the immunosuppressive actions of BMDMy are driven by phenotypically and epigenetically reprogrammed monocytes. Adoptive transfer of MEx-educated BMDMy, but not naive BMDMy, restored alveolar architecture, blunted fibrosis and pulmonary vascular remodeling, and improved exercise capacity. Conclusions: MEx ameliorate hyperoxia-induced neonatal lung injury though epigenetic and phenotypic reprogramming of myeloid cells.

Intratracheal transplantation of trophoblast stem cells attenuates acute lung injury in mice.

BACKGROUND: Acute lung injury (ALI) is a common lung disorder that affects millions of people every year. The infiltration of inflammatory cells into the lungs and death of the alveolar epithelial cells are key factors to trigger a pathological cascade. Trophoblast stem cells (TSCs) are immune privileged, and demonstrate the capability of self-renewal and multipotency with differentiation into three germ layers. We hypothesized that intratracheal transplantation of TSCs may alleviate ALI. METHODS: ALI was induced by intratracheal delivery of bleomycin (BLM) in mice. After exposure to BLM, pre-labeled TSCs or fibroblasts (FBs) were intratracheally administered into the lungs. Analyses of the lungs were performed for inflammatory infiltrates, cell apoptosis, and engraftment of TSCs. Pro-inflammatory cytokines/chemokines of lung tissue and in bronchoalveolar lavage fluid (BALF) were also assessed. RESULTS: The lungs displayed a reduction in cellularity, with decreased CD45+ cells, and less thickening of the alveolar walls in ALI mice that received TSCs compared with ALI mice receiving PBS or FBs. TSCs decreased infiltration of neutrophils and macrophages, and the expression of interleukin (IL) 6, monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) in the injured lungs. The levels of inflammatory cytokines in BALF, particularly IL-6, were decreased in ALI mice receiving TSCs, compared to ALI mice that received PBS or FBs. TSCs also significantly reduced BLM-induced apoptosis of alveolar epithelial cells in vitro and in vivo. Transplanted TSCs integrated into the alveolar walls and expressed aquaporin 5 and prosurfactant protein C, markers for alveolar epithelial type I and II cells, respectively. CONCLUSION: Intratracheal transplantation of TSCs into the lungs of mice after acute exposure to BLM reduced pulmonary inflammation and cell death. Furthermore, TSCs engrafted into the alveolar walls to form alveolar epithelial type I and II cells. These data support the use of TSCs for the treatment of ALI.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Restore Thymic Architecture and T Cell Function Disrupted by Neonatal Hyperoxia.

Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most notably bronchopulmonary dysplasia (BPD), a disease of disrupted lung development. The effects of high oxygen exposure on other developing organs of the infant, as well as the possible impact such disrupted development may have on later life remain poorly understood. Using a neonatal mouse model to investigate the effects of hyperoxia on the immature immune system we observed a dramatic involution of the thymic medulla, and this lesion was associated with disrupted FoxP3+ regulatory T cell generation and T cell autoreactivity. Significantly, administration of mesenchymal stromal cell-derived extracellular vesicles (MEx) restored thymic medullary architecture and physiological thymocyte profiles. Using single cell transcriptomics, we further demonstrated preferential impact of MEx treatment on the thymic medullary antigen presentation axis, as evidenced by enrichment of antigen presentation and antioxidative-stress related genes in dendritic cells (DCs) and medullary epithelial cells (mTECs). Our study demonstrates that MEx treatment represents a promising restorative therapeutic approach for oxygen-induced thymic injury, thus promoting normal development of both central tolerance and adaptive immunity.

Therapeutic Effects of Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles in Oxygen-Induced Multi-Organ Disease: A Developmental Perspective.

Despite major advances in neonatal intensive care, infants born at extremely low birth weight still face an increased risk for chronic illness that may persist into adulthood. Pulmonary, retinal, and neurocognitive morbidities associated with preterm birth remain widespread despite interventions designed to minimize organ dysfunction. The design of therapeutic applications for preterm pathologies sharing common underlying triggers, such as fluctuations in oxygen supply or in the inflammatory state, requires alternative strategies that promote anti-inflammatory, pro-angiogenic, and trophic activities-ideally as a unitary treatment. Mesenchymal stem/stromal cell-derived extracellular vesicles (MEx) possess such inherent advantages, and they represent a most promising treatment candidate, as they have been shown to contribute to immunomodulation, homeostasis, and tissue regeneration. Current pre-clinical studies into the MEx mechanism of action are focusing on their restorative capability in the context of preterm birth-related pathologies, albeit not always with a multisystemic focus. This perspective will discuss the pathogenic mechanisms underlying the multisystemic lesions resulting from early-life disruption of normal physiology triggered by high oxygen exposures and pro-inflammatory conditions and introduce the application of MEx as immunomodulators and growth-promoting mediators for multisystem therapy.

Mesenchymal stromal cell-derived extracellular vesicle therapy prevents preeclamptic physiology through intrauterine immunomodulation†.

Human umbilical cord-derived mesenchymal stromal cells (MSCs) are a widely recognized treatment modality for a variety of preclinical disease models and have been transitioned to human clinical trials. We have previously shown in neonatal lung disease that the therapeutic capacity of MSCs is conferred by their secreted extracellular vesicles (MEx), which function primarily through immunomodulation. We hypothesize that MEx have significant therapeutic potential pertinent to immune-mediated gestational diseases. Of particular interest is early-onset preeclampsia, which can be caused by alterations of the maternal intrauterine immune environment. Using a heme-oxygenase-1 null mouse model of pregnancy loss with preeclampsia-like features, we examined the preventative effects of maternal MEx treatment early in pregnancy. Heme oxygenase-1 null females (Hmox1-/-) or wild-type control females were bred in homozygous matings followed by evaluation of maternal and fetal parameters. A single dose of MEx was administered intravenously on gestational day (GD)1 to Hmox1-/- females (Hmox1-/- MEx). Compared with untreated Hmox1-/- females, Hmox1-/- MEx-treated pregnancies showed significant improvement in fetal loss, intrauterine growth restriction, placental spiral artery modification, and maternal preeclamptic stigmata. Biodistribution studies demonstrated that MEx localize to a subset of cells in the preimplantation uterus. Further, mass cytometric (CyTOF) evaluation of utero-placental leukocytes in Hmox1-/- MEx versus untreated pregnancies showed alteration in the abundance, surface marker repertoire, and cytokine profiles of multiple immune populations. Our data demonstrate the therapeutic potential of MEx to optimize the intrauterine immune environment and prevent maternal and fetal sequelae of preeclamptic disease.

Mesenchymal stromal cell-derived small extracellular vesicles restore lung architecture and improve exercise capacity in a model of neonatal hyperoxia-induced lung injury.

Early administration of mesenchymal stromal cell (MSC)-derived small extracellular vesicles (MEx) has shown considerable promise in experimental models of bronchopulmonary dysplasia (BPD). However, the ability of MEx to reverse the long-term pulmonary complications associated with established BPD remains unknown. In this study, MEx were isolated from media conditioned by human Wharton's Jelly-derived MSC cultures. Newborn mice (FVB strain) were exposed to hyperoxia (HYRX (75% O2)) before returning to room air at postnatal day 14 (PN14). Following prolonged HYRX-exposure, animals received a single MEx dose at PN18 or serial MEx treatments at PN18-39 ("late" intervention). This group was compared to animals that received an early single MEx dose at PN4 ("early" intervention). Animals were harvested at PN28 or 60 for assessment of pulmonary parameters. We found that early and late MEx interventions effectively ameliorated core features of HYRX-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. Exercise capacity testing and assessment of pulmonary hypertension (PH) showed functional improvements following both early and late MEx interventions. In conclusion, delivery of MEx following prolonged HYRX-exposure improves core features of experimental BPD, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating PH and improving exercise capacity. Taken together, delivery of MEx may not only be effective in the immediate neonatal period to prevent the development of BPD but may provide beneficial effects for the management and potentially the reversal of cardiorespiratory complications in infants and children with established BPD.

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Heme oxygenase-1 dampens the macrophage sterile inflammasome response and regulates its components in the hypoxic lung.

Exposure to hypoxia causes an inflammatory reaction in the mouse lung, and this response can be modulated by overexpressing the hypoxia-inducible stress-response enzyme, heme oxygenase-1 (HO-1). We hypothesized that the inflammasome activity may be a central pathway by which HO-1 controls pulmonary inflammation following alveolar hypoxia. Therefore, we investigated whether HO-1 controls inflammasome activation by altering its expression in macrophages primed with classic NOD-like receptor containing a pyrin domain 3 (NLRP3) inducers, and in murine lungs lacking HO-1 and exposed to acute hypoxia. We found that lack of HO-1 activated lipopolysaccharide (LPS) and ATP-treated bone marrow-derived macrophages, causing an increase in secreted levels of cleaved interleukin (IL)-1B, IL-18, and caspase-1, markers of increased inflammasome activity, whereas HO-1 overexpression suppressed IL-1B, NLRP3, and IL-18. The production of cleaved IL-1B and the activation of caspase-1 in LPS- and ATP-primed macrophages were inhibited by hemin, an HO-1 inducer, and two HO-1 enzymatic products [bilirubin and carbon monoxide (CO)]. Exposure of mice to hypoxia induced the expression of several inflammasome mRNA components (IL-1B, Nlrp3, and caspase-1), and this was further augmented by HO-1 deficiency. This pronounced inflammasome activation was detected as increased protein levels of apoptosis-associated speck-like protein containing a COOH-terminal caspase recruitment domain, IL-18, procaspase-1, and cleaved caspase-1 in the lungs of hypoxic mice. Systemically, Hmox1-deficient mice showed increased basal levels of IL-18 that were further increased after 48 h of hypoxic exposure. Taken together, these finding point to a pivotal role for HO-1 in the control of baseline and hypoxic inflammasome signaling, perhaps through the antioxidant properties of bilirubin and CO's pleiotropic effects.

Mesenchymal stromal cell exosomes prevent and revert experimental pulmonary fibrosis through modulation of monocyte phenotypes.

Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF). The aim of this study was to test the therapeutic effects of extracellular vesicles produced by human BM MSCs (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate mechanisms of action. Adult C57BL/6 mice were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently, or for reversal models, at day 7 or 21. Experimental groups were assessed at day 7, 14, or 28. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented or reversed by MEx treatment. MEx modulated lung macrophage phenotypes, shifting the proportions of lung proinflammatory/classical and nonclassical monocytes and alveolar macrophages toward the monocyte/macrophage profiles of control mice. A parallel immunomodulatory effect was demonstrated in the BM. Notably, transplantation of MEx-preconditioned BM-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation. Proteomic analysis revealed that MEx therapy promotes an immunoregulatory, antiinflammatory monocyte phenotype. We conclude that MEx prevent and revert core features of bleomycin-induced pulmonary fibrosis and that the beneficial actions of MEx may be mediated via systemic modulation of monocyte phenotypes.

Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications.

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation.

Macrophage Immunomodulation: The Gatekeeper for Mesenchymal Stem Cell Derived-Exosomes in Pulmonary Arterial Hypertension?

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of the pulmonary arteries, increased pulmonary infiltrates, loss of vascular cross-sectional area, and elevated pulmonary vascular resistance. Despite recent advances in the management of PAH, there is a pressing need for the development of new tools to effectively treat and reduce the risk of further complications. Dysregulated immunity underlies the development of PAH, and macrophages orchestrate both the initiation and resolution of pulmonary inflammation, thus, manipulation of lung macrophage function represents an attractive target for emerging immunomodulatory therapies, including cell-based approaches. Indeed, mesenchymal stem cell (MSC)-based therapies have shown promise, effectively modulating the macrophage fulcrum to favor an anti-inflammatory, pro-resolving phenotype, which is associated with both histological and functional benefits in preclinical models of pulmonary hypertension (PH). The complex interplay between immune system homeostasis and MSCs remains incompletely understood. Here, we highlight the importance of macrophage function in models of PH and summarize the development of MSC-based therapies, focusing on the significance of MSC exosomes (MEx) and the immunomodulatory and homeostatic mechanisms by which such therapies may afford their beneficial effects.