Abstract Background The variable clinical outcomes of mesenchymal stromal cell (MSC)-based therapy in acute respiratory distress syndrome (ARDS) are attributed to a variety of factors, including host microenvironmental factors. Interleukin-1β (IL-1β) has been linked to the development and progression of ARDS, and we have previously found that IL-1β could be used to predict MSC activation in vitro. However, the exact mechanisms through which IL-1β alters the MSC function and its interaction with the host immune cells remains unknown. Therefore, the aim of this study was to assess how IL-1β alters MSC function, with a specific focus on MSC-neutrophil interaction. Methods Human bone marrow-derived MSCs were exposed to 20 ng/ml IL-1β for 1 or 24 h. Following exposure, MSCs were analyzed using bulk RNA sequencing and key secretome proteins were measured in their conditioned medium. A transwell culture system was used to evaluate the neutrophil recruitment capacity of IL-1β-exposed MSCs, with or without NF-kB inhibition. MSCs exposed to serum free medium were used as controls in all experiments. Results The sequencing data revealed that genes involved in response to biotic stimuli and immune response were altered in MSCs exposed to IL-1β compared to control cells. In particular, genes essential for neutrophil recruitment were significantly upregulated after IL-1β exposure. The functional in vitro studies further validated these results, demonstrating that MSCs exposed to IL-1β had a significantly higher neutrophil recruitment capacity compared to unstimulated MSCs. Finally, inhibition of the NF-kB pathway resulted in a significant decrease of the MSC’s capacity to recruit neutrophils to levels similar as to the unstimulated control MSCs. Conclusion These data provide mechanistic insight into how inflammatory factors present in the host microenvironment might affect the interaction between MSCs and immune cells. This further highlights the need to understand the MSC mode of action, and to map out how the MSC fate might change in different host environments after administration.

Background and aims Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and their extracellular vesicles (EVs) reduce lung inflammation and fibrosis in a variety of model systems, including in a Cystic Fibrosis (CF) mouse model. Many components of MSC-derived EVs, including cytokines, antimicrobial peptides, and miRNAs have been implicated in their anti-inflammatory effects. However, a major gap in our knowledge of using MSC as a therapeutic intervention for people with CF (pwCF) is whether the CF airway environment compromises miRNA cargo in hBM-MSC-derived EVs. Methods To assess this, hBM-MSCs were exposed to cell culture media (control) or to bronchoalveolar lavage fluid (BALF) obtained from pwCF or healthy controls (HC) and compositional analysis of EV miRNA content was conducted. Results Thirteen miRNAs (each ≥1% of the total miRNA content) were identified that collectively account for ∼70% of the miRNA content of EVs. These miRNAs were remarkably stable across treatments. To infer potential therapeutic effects, we identified predicted gene targets of these miRNAs and performed pathway enrichment analysis. Gene pathway analysis revealed that many of the 13 miRNAs are predicted to inhibit TLR signaling, NF-κB activation, TGF-β-mediated fibrosis, and cytokine production. Conclusions These results indicate that miRNAs secreted by hBM-MSCs in EVs may contribute to the observed anti-inflammatory and anti-fibrotic effects in experimental models and that exposure to CF BALF does not significantly diminish the abundance of the 13 miRNAs.

The in vivo fate of mesenchymal stromal cells (MSCs), including their clearance, interaction with host tissues, and persistence, remains incompletely understood following systemic or local clinical administration to patients. Although immune-mediated clearance mechanisms, such as triggering of the instant blood-mediated inflammatory reaction, activation of coagulation and complement pathways, apoptosis and efferocytosis have been identified, their contributions to MSC function and efficacy are still under investigation. To address these knowledge gaps, an international panel of experts in MSC biology and clinical regenerative medicine convened to assess current evidence and define key unanswered questions. Discussions were structured around three thematic domains: (i) biodistribution and mechanisms of action following systemic delivery; (ii) biological implications of local or depot-based administration and (iii) the dynamics of MSC persistence and clearance in vivo. A major focus was on the role of MSC apoptosis and its immunological consequences, particularly interactions between apoptotic MSCs, phagocytes and endothelial barriers. This perspective highlights the most urgent research questions identified during the meeting and in follow-up discussions and proposes experimental strategies to move beyond traditional cell tracking toward interrogating functional persistence, immune modulation and delivery context. Addressing these gaps will deepen our understanding of MSC behavior in vivo and guide the development of safer, more predictable and more effective MSC-based interventions.

The acute respiratory distress syndrome (ARDS) inflammatory environment alters mesenchymal stromal cell (MSC) gene and protein expression but effects on microRNA (miRNA) content of MSC-extracellular vesicle (EVs) remain unknown. To assess this, sequencing analysis of EV-miRNAs prepared from human bone marrow-derived MSCs (hMSCs) exposed ex vivo to bronchoalveolar lavage fluid (BALF) from ARDS patients or healthy volunteers (HV) identified a number of differentially expressed miRNAs. Discriminant, differential expression, and functional enrichment analyses identified 14 miRNAs significantly changed following ARDS versus HV BALF exposure. Network analysis showed 4 (miR-760, miR-3175, miR-885-3p, and miR-766-3p) of the 14 EV-miRNAs formed a regulatory “hub”, suggesting co-targeting of specific gene pathways. In silico prediction identified a number of pathways important in lung injury. Two miRNAs involved in regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), miRNA-145-5p and miRNA-138-5p, were also significantly increased in ARDS BALF-exposed hMSCs EVs. Functionally, EVs from hMSCs exposed to either ARDS or HV BALF had differential effects on CFTR Cl- secretion by cultured primary human bronchial epithelial cells, an effect predicted to reduce mucociliary clearance. The potential clinical impact of these finding highlights the need for further studies assessing the role of hMSC-EV miRNAs in regulating lung inflammation and mucociliary clearance.

The in vivo fate of mesenchymal stromal cells (MSCs), including their clearance, interaction with host tissues, and persistence, remains incompletely understood following systemic or local clinical administration to patients. Although immune-mediated clearance mechanisms, such as triggering of the instant blood-mediated inflammatory reaction (IBMIR), coagulation and complement pathways activation, apoptosis, and efferocytosis have been identified, their contributions to MSC function and efficacy are still under investigation. To address these knowledge gaps, an international panel of global experts in MSC biology and clinical regenerative medicine convened to assess current evidence and define key unanswered questions. Discussions were structured around three thematic domains: (1) biodistribution and mechanisms following systemic delivery; (2) biological implications of local or depot-based administration, and (3) the dynamics of MSC persistence and clearance in vivo. A major focus was on the role of MSC apoptosis and its immunological consequences, particularly interactions between apoptotic MSCs, phagocytes, and endothelial barriers. This perspective highlights the most urgent research questions identified during the meeting and in follow-up discussions and proposes experimental strategies to move beyond traditional cell tracking toward interrogating functional persistence, immune modulation, and delivery context. Addressing these gaps will deepen our understanding of MSCs in vivo and guide the development of safer, predictable, and effective MSC-based interventions.

For many severe lung diseases, non-invasive biomarkers from imaging could improve early detection of lung injury or disease onset, establish a diagnosis, or help follow-up disease progression and treatment strategies. Imaging of the thorax and lung is challenging due to its size, respiration movement, transferred cardiac pulsation, vast density range and gravitation sensitivity. However, there is extensive ongoing research in this fast-evolving field. Recent improvements in spatial imaging have allowed us to study the three-dimensional structure of the lung, providing both spatial architecture and transcriptomic information at single-cell resolution. This fast progression, however, comes with several challenges, including significant image file storage and network capacity issues, increased costs, data processing and analysis, the role of artificial intelligence and machine learning, and mechanisms to combine several modalities. In this review, we provide an overview of advances and current issues in the field of spatial lung imaging.

Mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) have emerged as innovative therapeutic agents for the treatment of sepsis and acute respiratory distress syndrome (ARDS). Although their potential remains undisputed in pre-clinical models, this has yet to be translated to the clinic. In this review, we focused on the role of microRNAs contained in MSC-derived EVs, the EV microRNAome, and their potential contribution to therapeutic mechanisms of action. The evidence that miRNA transfer in MSC-derived EVs has a role in the overall therapeutic effects is compelling. However, several questions remain regarding how to reconcile the stochiometric issue of the low copy numbers of the miRNAs present in the EV particles, how different miRNAs delivered simultaneously interact with their targets within recipient cells, and the best miRNA or combination of miRNAs to use as therapy, potency markers, and biomarkers of efficacy in the clinic. Here, we offer a molecular genetics and systems biology perspective on the function of EV microRNAs, their contribution to mechanisms of action, and their therapeutic potential.

Purpose of Review Despite no general conclusions regarding the therapeutic effect of MSCs on virus-induced acute lung injury in pre-clinical studies, a significant number of clinical trials using MSC-based treatment for COVID-19-associated ARDS were initiated during the global pandemic. Here, we aimed to discuss differences and similarities in clinical trials using MSC-based treatments for classical ARDS and COVID-19-associated ARDS and to raise some future perspectives. Recent Findings Several pre-clinical studies have demonstrated that MSC treatment may not be a good treatment option for virus infections because MSCs themselves are susceptible to the virus. However, MSCs lack expression of the angiotensin-converting enzyme 2 (ACE2) receptor, suggesting that MSCs are not likely to be infected by the COVID-19 virus. Interestingly, recent meta-analyses demonstrated that an improved survival rate in patients with COVID-19-associated ARDS treated with MSCs was obtained in 24 out of 26 completed clinical trials. Summary This review provides comparative perspectives on MSC-based therapy for COVID-19-associated ARDS and classical ARDS.

In this review, the Basic and Translational Science Assembly of the European Respiratory Society provides an overview of the 2022 International Congress highlights. We discuss the consequences of respiratory events from birth until old age regarding climate change related alterations in air quality due to pollution caused by increased ozone, pollen, wildfires and fuel combustion as well as the increasing presence of microplastic and microfibres. Early life events such as the effect of hyperoxia in the context of bronchopulmonary dysplasia and crucial effects of the intrauterine environment in the context of pre-eclampsia were discussed. The Human Lung Cell Atlas (HLCA) was put forward as a new point of reference for healthy human lungs. The combination of single-cell RNA sequencing and spatial data in the HLCA has enabled the discovery of new cell types/states and niches, and served as a platform that facilitates further investigation of mechanistic perturbations. The role of cell death modalities in regulating the onset and progression of chronic lung diseases and its potential as a therapeutic target was also discussed. Translational studies identified novel therapeutic targets and immunoregulatory mechanisms in asthma. Lastly, it was highlighted that the choice of regenerative therapy depends on disease severity, ranging from transplantation to cell therapies and regenerative pharmacology.

Introduction: Mesenchymal stromal cells (MSC)-based therapies have advanced into clinical settings in several diseases including ARDS. Yet, no significant outcome has been demonstrated. Increasing evidence suggests that MSCs alter their therapeutic functions when exposed to external factors including the local inflammatory environments. ARDS is a clinical syndrome that includes at least 2 phenotypes (hyper- and hypo-inflammation) with very different clinical pathways. However, it is still unknown if any of the phenotypes are more beneficial for MSC-based therapies, or if the underlaying biology of these phenotypes alter MSC functions. Therefore, the aim of this study was to test the effect of ARDS disease-relevant cytokines on human bone marrow-derived MSC function (hMSCs). Methods: hMSCs were exposed to disease-relevant cytokines for 1 or 24 hours. Gene and protein expression will be analyzed on exposed hMSCs and compared to control cells. Conditioned medium was collected for determination of the secretome profiles, immune-regulatory functions, and cytotoxicity analyzes. Results: Preliminary data suggests that hMSCs exposed to cytokines highly abundant in ARDS patients with a hyperinflammatory sub-phenotype had increased metabolic activity, increased proliferation rate, and altered secretome profile compared to hMSCs exposed to medium alone. Conditioned medium was collected and functional immunoregulatory studies are currently ongoing. Conclusions: A better understanding on how the microenvironment in patients with different ARDS sub-phenotypes influence hMSCs functions will help us to better design clinical trials and increase the success rate of MSC-based therapies of ARDS.

RationaleA better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming.MethodsHuman monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic.ResultsEVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model.ConclusionmiR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.

Current asthma therapies focus on reducing symptoms but fail to restore existing structural damage. Mesenchymal stromal cell (MSC) administration can ameliorate airway inflammation and reverse airway remodeling. However, differences in patient disease microenvironments seem to influence MSC therapeutic effects. A polymorphic CATT tetranucleotide repeat at position 794 of the human macrophage migration inhibitory factor (hMIF) gene has been associated with increased susceptibility to and severity of asthma. We investigated the efficacy of human MSCs in high- vs. low-hMIF environments and the impact of MIF pre-licensing of MSCs using humanized MIF mice in a clinically relevant house dust mite (HDM) model of allergic asthma. MSCs significantly attenuated airway inflammation and airway remodeling in high-MIF-expressing CATT7 mice but not in CATT5 or wild-type littermates. Differences in efficacy were correlated with increased MSC retention in the lungs of CATT7 mice. MIF licensing potentiated MSC anti-inflammatory effects at a previously ineffective dose. Mechanistically, MIF binding to CD74 expressed on MSCs leads to upregulation of cyclooxygenase 2 (COX-2) expression. Blockade of CD74 or COX-2 function in MSCs prior to administration attenuated the efficacy of MIF-licensed MSCs in vivo. These findings suggest that MSC administration may be more efficacious in severe asthma patients with high MIF genotypes (CATT6/7/8).

Mesenchymal stromal cells (MSCs) were identified more than 50 years ago, and research advances have promoted the translation of pre-clinical studies into clinical settings in several diseases. However, we are only starting to uncover the local factors that regulate cell phenotype, cell function, and cell viability across tissues following administration in different diseases. Advances in pre-clinical and translational studies suggest that the host environment, especially inflammatory active environments, plays a significant role in directing the infused MSCs towards different phenotypes with different functions. This can significantly effect their therapeutic efficacy. One way to study this interaction between the host environment and the infused cells is to expose MSCs ex vivo to patient samples such as serum or bronchoalveolar lavage fluid. Using this approach, it has been demonstrated that MSCs are very sensitive to different host factors such as pathogens, inflammatory cytokines, and extra cellular matrix properties. By understanding how different local host factors effect MSC function it will open possibilities to select specific patient sub-groups that are more likely to respond to this type of treatment and will also open possibilities to prime the local host environment to increase viability and to enrich for a specific MSC phenotype. Here, we aim to review the current understanding of the interaction of MSCs with the host microenvironment. To narrow the scope of this mini review, the focus will be on the pulmonary microenvironment, with a specific focus on the diseases acute respiratory distress syndrome (ARDS) and cystic fibrosis (CF).

Background A subset of individuals with allergic asthma develops a late phase response (LPR) to inhaled allergens, which is characterized by a prolonged airway obstruction, airway inflammation and airway hyperresponsiveness. The aim of this study was to identify changes in the plasma proteome and circulating hematopoietic progenitor cells associated with the LPR following inhaled allergen challenge. Methods Serial plasma samples from asthmatics undergoing inhaled allergen challenge were analyzed by mass spectrometry and immunosorbent assays. Peripheral blood mononuclear cells were analyzed by flow cytometry. Mass spectrometry data were analyzed using a linear regression to model the relationship between airway obstruction during the LPR and plasma proteome changes. Data from immunosorbent assays were analyzed using linear mixed models. Results Out of 396 proteins quantified in plasma, 150 showed a statistically significant change 23 h post allergen challenge. Among the most upregulated proteins were three protease inhibitors: alpha-1-antitrypsin, alpha-1-antichymotrypsin and plasma serine protease inhibitor. Altered levels of 13 proteins were associated with the LPR, including increased factor XIII A and decreased von Willebrand factor. No relationship was found between the LPR and changes in the proportions of classical, intermediate, and non-classical monocytes. Conclusions Allergic reactions to inhaled allergens in asthmatic subjects were associated with changes in a large proportion of the measured plasma proteome, whereof protease inhibitors showed the largest changes, likely to influence the inflammatory response. Many of the proteins altered in relation to the LPR are associated with coagulation, highlighting potential mechanistic targets for future treatments of type-2 asthma.

Dipeptidyl peptidase 4 (DPP4) has been proposed as a marker for activated fibroblasts in fibrotic disease. We aimed to investigate whether a profibrotic DPP4 phenotype is present in lung tissue from patients with idiopathic pulmonary fibrosis (IPF). The presence of DPP4+ fibroblasts in normal and IPF lung tissue was investigated using flow cytometry and immunohistology. In addition, the involvement of DPP4 in fibroblast activation was examined in vitro, using CRISPR/Cas9 mediated genetic inactivation to generate primary DPP4 knockout lung fibroblasts. We observed a reduced frequency of primary DPP4+ fibroblasts in IPF tissue using flow cytometry, and an absence of DPP4+ fibroblasts in pathohistological features of IPF. The in vivo observations were supported by results in vitro showing a decreased expression of DPP4 on normal and IPF fibroblasts after profibrotic stimuli (transforming growth factor β) and no effect on the expression of activation markers (α-smooth muscle actin, collagen I and connective tissue growth factor) upon knockout of DPP4 in lung fibroblasts with or without activation with profibrotic stimuli.

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs.

Mesenchymal cells are important components of specified niches in the lung, and can mediate a wide range of processes including tissue regeneration and repair. Dysregulation of these processes can lead to improper remodeling of tissue as observed in several lung diseases. The mesenchymal cells responsible remain poorly described, partially due to the heterogenic nature of the mesenchymal compartment and the absence of appropriate markers. Here, we describe that CD105+CD90+ mesenchymal cells can be divided into two populations based on their expression of CD13/aminopeptidase N (CD105+CD90+CD13− and CD105+CD90+CD13+). By prospective isolation using FACS, we show that both these populations give rise to clonogenic fibroblast-like cells, but with an increased clonogenic and proliferative capacity of CD105+CD90+CD13+ cells. Transcriptomic and spatial analysis pinpoints an adventitial fibroblast subset as the origin of CD105+CD90+CD13+ clonogenic mesenchymal cells in human lung.